Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 24 2015

3

INTRODUCTION

4

INTRODUCTION

WHAT WHERE WHEN WHY HOW

5

INTRODUCTION

miRNAmicroRNA

WHAT

RNA interference (RNAi)Short non-coding RNA (20-22 nt)RNA silencing pathwaymiRNA-miRNA duplex (miRNA is the antisense or guide strand and miRNA is the sense or passenger strand)

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

6

INTRODUCTION

microRNA

WHERE

DGCR8 microprocessor complex subunit (DiGeorge syndrome chromosomal [or critical] region 8)Drosha and dicer RNase III enzyme

pri-miRNA = primary transcript miRNA

pre-miRNA = precursor miRNA

RNA-induced silencing complex (RISC)

Transactivating response RNA-binding protein (TRBP)

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

7

INTRODUCTION

microRNA

WHY

Apoptosis

Proliferation

Differentiation and maturation

DESEASE

MUTATION

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

3

INTRODUCTION

4

INTRODUCTION

WHAT WHERE WHEN WHY HOW

5

INTRODUCTION

miRNAmicroRNA

WHAT

RNA interference (RNAi)Short non-coding RNA (20-22 nt)RNA silencing pathwaymiRNA-miRNA duplex (miRNA is the antisense or guide strand and miRNA is the sense or passenger strand)

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

6

INTRODUCTION

microRNA

WHERE

DGCR8 microprocessor complex subunit (DiGeorge syndrome chromosomal [or critical] region 8)Drosha and dicer RNase III enzyme

pri-miRNA = primary transcript miRNA

pre-miRNA = precursor miRNA

RNA-induced silencing complex (RISC)

Transactivating response RNA-binding protein (TRBP)

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

7

INTRODUCTION

microRNA

WHY

Apoptosis

Proliferation

Differentiation and maturation

DESEASE

MUTATION

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

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copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

4

INTRODUCTION

WHAT WHERE WHEN WHY HOW

5

INTRODUCTION

miRNAmicroRNA

WHAT

RNA interference (RNAi)Short non-coding RNA (20-22 nt)RNA silencing pathwaymiRNA-miRNA duplex (miRNA is the antisense or guide strand and miRNA is the sense or passenger strand)

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

6

INTRODUCTION

microRNA

WHERE

DGCR8 microprocessor complex subunit (DiGeorge syndrome chromosomal [or critical] region 8)Drosha and dicer RNase III enzyme

pri-miRNA = primary transcript miRNA

pre-miRNA = precursor miRNA

RNA-induced silencing complex (RISC)

Transactivating response RNA-binding protein (TRBP)

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

7

INTRODUCTION

microRNA

WHY

Apoptosis

Proliferation

Differentiation and maturation

DESEASE

MUTATION

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

5

INTRODUCTION

miRNAmicroRNA

WHAT

RNA interference (RNAi)Short non-coding RNA (20-22 nt)RNA silencing pathwaymiRNA-miRNA duplex (miRNA is the antisense or guide strand and miRNA is the sense or passenger strand)

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

6

INTRODUCTION

microRNA

WHERE

DGCR8 microprocessor complex subunit (DiGeorge syndrome chromosomal [or critical] region 8)Drosha and dicer RNase III enzyme

pri-miRNA = primary transcript miRNA

pre-miRNA = precursor miRNA

RNA-induced silencing complex (RISC)

Transactivating response RNA-binding protein (TRBP)

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

7

INTRODUCTION

microRNA

WHY

Apoptosis

Proliferation

Differentiation and maturation

DESEASE

MUTATION

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

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copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

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copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

6

INTRODUCTION

microRNA

WHERE

DGCR8 microprocessor complex subunit (DiGeorge syndrome chromosomal [or critical] region 8)Drosha and dicer RNase III enzyme

pri-miRNA = primary transcript miRNA

pre-miRNA = precursor miRNA

RNA-induced silencing complex (RISC)

Transactivating response RNA-binding protein (TRBP)

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

7

INTRODUCTION

microRNA

WHY

Apoptosis

Proliferation

Differentiation and maturation

DESEASE

MUTATION

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

7

INTRODUCTION

microRNA

WHY

Apoptosis

Proliferation

Differentiation and maturation

DESEASE

MUTATION

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

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copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

8

INTRODUCTIONWHEN

microRNA

Principles of microRNAndashmRNA interactions

Nucleotide 1 A Seed region (Nucleotides 2-8) Perfect base pairing Nucleotide 9 A or U Nucleotide 13-16 Good base pairing

miRNA

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

Internal ribosomal entry site(IRES) An RNA element usually present in the 5 UTR that allows m7G-cap-independent association of ribosome with mRNA

ApppN capAn unmethylated cap analogue that is not bound by eIF4E The mRNAs with an artificially introduced ApppN cap instead of a physiological m7GpppN cap are translated inefficiently

interaction with an internal ribosome entry site (IRES)42 Joining of the 60S subunit at the AUG codon precedes the elongation phase of translation

Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabili-zation or translational repression whether the latter occurs at the initiation or post-initiation step (or both) remains a matter of debate Several recently published papers provide important mechanistic insights into the repression-at-the-initiation step giving extra credence to this model

Repression at the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G-capped mRNAs but not of mRNAs containing an IRES or a non-functional ApppN

cap is repressed by miRNAs4344 As in numerous subse-quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with bi-cistronic mRNAs In these experi-ments the activity of the first cap-dependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous let-7 miRNA43 (FIG 1) Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let-7-binding sites or that were repressed by AGO2 (artificially tethered to the 3 UTR) showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA43 Likewise the amino-acid- starvation-induced release of endogenous cationic amino acid transporter 1 (CAT1) mRNA from repression that was mediated by the miRNA miR-122 was accompa-nied by a more effective recruitment of CAT1 mRNA to polysomes in human hepatoma cells45

There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EndasheIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 40S initiation complex46ndash48 Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et al49 recently reported that the central domain of AGO proteins contains limited sequence homology to the cap-binding region of eIF4E Importantly the similarity includes two aromatic resi-dues (FIG 2) which are crucial for cap binding in eIF4E and other cap-binding proteins4950 Mutations of one or both aromatic amino acids in AGO2 to valine but signif-icantly not to other aromatic amino acids prevented the interaction with m7GTPndashSepharose and abolished the ability of AGO2 to repress translation when tethered to the mRNA 3 UTR These data indicate that AGO2 and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IRES-containing mRNAs49 The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust repres-sion27304351 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E49 would increase the likelihood of AGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNA-mediated repression in a physiological assay Additional evidence for example from cross-linking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure

Lessons from in vitro studies Four different cell-free extracts that recapitulate many features of the miRNA-mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repression52ndash55 the mRNAs containing

Box 2 | Principles of microRNAndashmRNA interactions

MicroRNAs (miRNAs) interact with their mRNA targets by base pairing In plants most miRNAs base pair to mRNAs with nearly perfect complementarity and induce mRNA degradation by an RNAi-like mechanism mdash the mRNA is cleaved endonucleolytically in the middle of the miRNAndashmRNA duplex29 By contrast with few exceptions metazoan miRNAs base pair with their targets imperfectly following a set of rules that have been identified by experimental and bioinformatics analyses30ndash34bull One rule for miRNAndashtarget base paring is perfect and contiguous base pairing of

miRNA nucleotides 2 to 8 representing the lsquoseedrsquo region (shown in dark red and green) which nucleates the miRNAndashmRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 of the miRNA and an A or U across position 9 (shown in yellow) improve the site efficiency although they do not need to base pair with miRNA nucleotides

bull Another rule is that bulges or mismatches must be present in the central region of the miRNAndashmRNA duplex precluding the Argonaute (AGO)-mediated endonucleolytic cleavage of mRNA

bull The third rule is that there must be reasonable complementarity to the miRNA 3 half to stabilize the interaction Mismatches and bulges are generally tolerated in this region although good base pairing particularly to residues 13ndash16 of the miRNA (shown in orange) becomes important when matching in the seed region is suboptimal3133

Other factors that can improve site efficacy include an AU-rich neighbourhood and for long 3 UTRs a position that is not too far away from the poly(A) tail or the termination codon these factors can make the 3 UTR regions less structured and hence more accessible to miRNP recognition3334129 Indeed accessibility of binding sites might have an important effect on miRNA-mediated repression130 Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131132 In addition combinations of sites can require a specific configuration (for example separation by a stretch of nucleotides of specific sequence and length) for efficient repression131 Usually miRNA-binding sites in metazoan mRNAs lie in the 3 UTR and are present in multiple copies Importantly multiple sites for the same or different miRNAs are generally required for effective repression30ndash34 When they are present close to each other (10ndash40 nucleotides apart) they tend to act cooperatively that is their effect exceeds that expected from the independent contributions of two single sites3033

NNNNNNN

Nature Reviews | Genetics

Bulge

ORF AAAAAA

gt15 nucleotides

lsquoSeedrsquoregion

Bulge

3 complementarity

816 13 1

A

NNNNNNNNNN

NNNNNNNNNNAU

NNNNNNNNN miRNA

R E V I E W S

NATURE REVIEWS | GENETICS VOLUME 9 | FEBRUARY 2008 | 105

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

9

INTRODUCTIONHOW

microRNA

Structural biology of microRNA for targeting

HOW

Stepwise mechanism

miRNA

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

Nature Reviews | Molecular Cell Biology

(A)n

Pri-miRNA

Processing

Maturation

Strand selectionRISC assembly

Initiation orelongation block Deadenylation

Transcription

Pre-miRNA

Drosha

DGCR8

m7G (A)n

Exportin 5

DicerTRBP

Ago

Ago

Ago

CCR4ndashNOT

P-bodies

Target repression

AGO1ndashAGO4

RISC

AAAAAORF

AAAAAORF

EIF4E Ribosome

Nucleus

Cytoplasm

biological processes might be prime candidates for miRNA-mediated regulation mdash might be more produc-tive In this Review we provide examples showing that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells Signalling complexes are indeed highly dynamic ephemeral and non-stoichiometric molecular ensembles which trans-late into well-established dose-dependent responses As such they are the ideal targets for the degree of quant-itative fluctuations imposed by miRNAs This might enable the multi-gene regulatory capacity of miRNAs to remodel the signalling landscape facilitating or oppos-ing the transmission of information to downstream effectors in an effective and timely manner20 TABLE 1 and Supplementary information S1 (table) provide a list of miRNAs targeting either positive or negative modulators of key signalling pathways In the first part of this Review we summarize some examples that relate the function of individual miRNAs to the regulation of cell signalling

miRNAs may also help to explain a paradox in evolution The core protein engines of developmental

signalling networks are highly conserved devices which can be traced back to the common ancestor of all Bilateria21ndash23 However the development of increasingly complex body plans obviously required a great degree of plasticity in the use of those pathways demanding the evolution of new layers of regulation Just like trans-cription factor binding sites 3 UTR sequences are not constrained by coding needs and can potentially diverge rapidly to co-opt beneficial miRNAndashtarget inter-actions and counter-select against deleterious pairs2425 Nevertheless although few new transcription factor families have arisen in animal evolution continuous emergence of new miRNA families has paralleled the increased complexities in body plans and organs242627 Thus miRNAs may represent ductile and fast-evolving tools which add sophisticated regulatory tiers to signal-ling pathways We discuss the logic of these networks in the second part of this Review In sum crosstalk between growth factor signalling and miRNAs may substantially contribute to our current understanding of miRNA biology

Box 1 | RNA biogenesis and mechanisms of action

MicroRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II Each pri-miRNA contains one or more hairpin structures that are recognized and processed by the microprocessor complex which consists of the RNase III type endonuclease Drosha and its partner DGCR8 (see the figure) The microprocessor complex generates a 70-nucleotide stem loop known as the precursor miRNA (pre-miRNA) which is actively exported to the cytoplasm by exportin 5

In the cytoplasm the pre-miRNA is recognized by Dicer another RNase III type endonuclease and TAR RNA-binding protein (TRBP also known as TARBP2) Dicer cleaves this precursor generating a 20-nucleotide mature miRNA duplex Generally only one strand is selected as the biologically active mature miRNA and the other strand is degraded The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which contains Argonaute (Ago) proteins and the single-stranded miRNA Mature miRNA allows the RISC to recognize target mRNAs through partial sequence complementarity with its target In particular perfect base pairing between the seed sequence of the miRNA (from the second to the eighth nucleotide) and the seed match sequences in the mRNA 3 UTR are crucial The RISC can inhibit the expression of the target mRNA through two main mechanisms that have several variations removal of the polyA tail (deadenylation) by fostering the activity of deadenylases (such as CCR4ndashNOT) followed by mRNA degradation and blockade of translation at the initiation step or at the elongation step for example by inhibiting eukaryotic initiation factor 4E (EIF4E) or causing ribosome stalling RISC-bound mRNA can be localized to sub-cytoplasmatic compartments known as P-bodies where they are reversibly stored or degraded

Figure is modified with permission from REF 104 Nature Reviews Genetics 2008 Macmillan Publishers Ltd All rights reserved m7G 7-methylguanosine cap ORF open reading frame

REVIEWS

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | APRIL 2010 | 253

copy 20 Macmillan Publishers Limited All rights reserved10

10

INTRODUCTION

To study structure of Argonaute 2 (Ago2) bound to guide RNA with

and without target RNAs

OBJECTIVE

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

11

INTRODUCTION

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

in which poly(A) tails are shortened in a distribu-tive manner without causing full mRNA degradation Only when the poly(A) tails are shortened to a certain length does the CCR4ndashNOT complex take over and deadenylate the mRNA in a processive manner com-mitting it to full decay3168 Consequently the activity of the PAN2ndashPAN3 complex may not be detectable unless the CCR4ndashNOT complex is inhibited and the length of the mRNA poly(A) tail is accurately determined Nevertheless depletion of PAN3 exacerbates the effects of NOT1 depletion25 indicating that PAN2ndashPAN3 participates in the degradation of miRNA reporters

Interaction of GW182 proteins with the CCR4ndashNOT complex The CCR4ndashNOT deadenylase complex has a central role in post-transcriptional mRNA regula-tion31 It catalyses the shortening of mRNA poly(A) tails and consequently causes or consolidates translational repression As mentioned above the complex couples deadenylation to decapping and 5ʹ-to-3ʹ exonucleolytic degradation by XRN1 therefore it can also lead to full degradation of the target mRNA in some cellular contexts31 In addition the CCR4ndashNOT complex has the remarkable ability to repress translation in the absence of mRNA deadenylation and decay263069ndash71 Accordingly in the context of the miRNA pathway the CCR4ndashNOT complex not only mediates deadenylation but is also involved in both the translational repression and the degradation of miRNA targets162124ndash27293070

The CCR4ndashNOT complex consists of several inde-pendent modules that dock with NOT1 the central scaf-fold subunit (FIG 4a) NOT1 features a modular domain organization (FIG 4a) consisting of separate α-helical domains which provide binding surfaces for the individ-ual modules A central domain of NOT1 mdash structurally related to the middle portion of eukaryotic translation initiation factor 4G (eIF4G) and thus termed the NOT1 MIF4G domain mdash is the docking site for the catalytic module which comprises two deadenylases namely CAF1 (or its paralogue POP2 also known as CNOT7 and CNOT8 respectively in humans) and CCR4A (or its paralogue CCR4B also known as CNOT6 and CNOT6L respectively in humans)317273 (FIG 4ab) The NOT1 MIF4G domain also serves as a binding platform for DDX6 (REFS 2930) (FIG 4cndashe) which functions as a translational repressor in addition to interacting with decapping factors such as EDC3 (REFS 29303274) Thus the NOT1 MIF4G domain coordinates the activities of the CCR4ndashNOT complex by providing binding sites for the factors that catalyse deadenylation translational repression and decapping29

Immediately downstream of the MIF4G domain NOT1 contains a CAF40NOT9-binding domain (CN9BD) which forms a stoichiometric complex with the highly conserved NOT9 subunit293071 This binary complex mediates binding to the GW182 proteins through tan-dem W-binding pockets present in the NOT9 subunit2930 (FIG 4afg) Similar to the structure of AGO2 (REF 59) the

Nature Reviews | Genetics

c H sapiens AGO2 PIWI domain

N MID PIWIPAZ L1 L2

V591

F587

P590

F659

Y698

Y654

F653

L694

E695

K660

20ndash25 Aring

W2W1

N-terminal domain

MID

PIWIPAZ

C-terminallobe

N-terminallobe

miRNA 5prime end

miRNA 3prime end

W1

W2

TargetmRNA

a H sapiens AGO2 b Complex of miRNA-bound H sapiens AGO2 and a target mRNA

Figure 2 | Structural insight into the interaction of AGO proteins with GW182 proteins a | Argonaute (AGO) proteins have four domains the amino-terminal domain the PIWIndashAGOndashZWILLE (PAZ) domain the MID domain and the PIWI domain The PAZ domain is connected to the N-terminal and MID domains by linker regions called L1 and L2 respectively Homo sapiens AGO2 is shown as a representative example of this family b | The structure of H sapiens AGO2 (green and grey) in complex with a microRNA (miRNA black) and a short piece of target mRNA (orange) (RCSB Protein Data Bank code 4W5O)61 highlights the position of the tryptophan (W) residues (red) bound to pockets on the surface of the PIWI domain c | Close-up view of the W-binding pockets (PDB code 4OLB)59 is shown Residues lining the pockets are shown as sticks and partially labelled for orientation The minimal distance between the two W residues is indicated C-terminal carboxy-terminal

REVIEWS

NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5

copy 2015 Macmillan Publishers Limited All rights reserved

PAZ domain A conserved nucleic-acid-binding structure that is found in members of the Dicer and Argonaute protein families

PIWI domain A conserved structure that is found in members of the Argonaute protein family It is structurally similar to ribonuclease H domains and in at least some cases has endoribonuclease activity

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

12

EXPERIMENTS

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

13

EXPERIMENTS

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

1 2 3

Protein Expression and Purification

Crystallization

Structure Determination

4

Analysis

(2-9 paired)(2-8 paired)(2-7 paired)

(2-8 paired long)

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

14

EXPERIMENTS

1 2 3

Protein Expression and Purification

4

Full length human Ago2 was cloned into pFastBac HT A for expression using the bac-2-bac (Invitrogen) baculovirus expression system and over-expressed in Sf9 cells

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

15

EXPERIMENTS

1 2 3

Crystallization

4

Crystals of guide-loaded Ago2 were grown using hanging drop vapor diffusion

Ago2-guide-target complexes were formed by the addition of 12 molar equivalents of target RNA at room temperature for 10 minutes Crystals of guide-loaded Ago2 bound to target RNA were grown using hanging drop vapor diffusion

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

16

EXPERIMENTS

1 2 3

Structure Determination

4

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

Ago2 crystal 4W5N 4W5O 4W5Q 4W5R 4W5T Target RNA guide only 2-9 paired 2-8 paired 2-8 paired (long) 2-7 paired Beamline SSRL 11-1 SSRL 12-2 SSRL 11-1 APS 24-ID-E SSRL 12-2 Space Group P1211 P1211 P1211 P1211 P1211 Unit Cell Dimensions A (Aring) 6312 5581 5550 5538 5564

B (Aring) 10887 11699 11665 11618 11647 C (Aring) 6807 6977 7007 6968 6982 α (deg) 9000 9000 9000 9000 9000 β (deg) 10693 9245 9226 9217 9234 γ (deg) 9000 9000 9000 9000 9000

Ago2 Molecules per ASU 1 1 1 1 1 Data Collection Wavelength (Aring) 097945 097950 097944 097918 097950 Resolution (Aring) 3898-290 3900-180 3888-310 11616-250 3882-250 (308-290) (184-180) (331-310) (264-250) (264-250) No Reflections Total 67638 276820 55568 113374 104468 Unique 19381 80397 16052 30428 29434 Completeness () 988 (988) 977 (920) 989 (989) 998 (999) 958 (976) Redundancy 35 (36) 34 (34) 35 (34) 37 (37) 35 (36) IσI 156 (26) 115 (22) 106 (25) 90 (19) 125 (33) Rmerge 70 (447) 55 (535) 94 (396) 141 (758) 74 (398) Rpim 68 (431) 51 (479) 90 (75) 85 (457) 46 (243) Refinement Resolution (Aring) 3898-290 3900-180 3529-310 6963-250 3529-250 R-freeR-factor 25332173 19651672 23251903 2341946 21511728 RMS Deviation Bond Distances (Aring) 0010 0007 0011 0006 0009 Bond Angles (deg) 1035 1142 1218 07820 1194 Number of Atoms Non-hydrogen protein 6461 6419 6439 6442 6471 Non-hydrogen RNA 355 595 572 406 527 Phenol 21 28 28 21 28

Isopropanol 0 8 0 8 0 Phosphate 0 5 0 0 0 Mg 1 3 3 2 2 Water 34 433 0 126 156

Ramachandran Plot Most Favored Regions 9383 9620 9395 9570 9611 Additionally Allowed 567 368 555 405 364 Generously Allowed 050 013 05 025 025

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

17

EXPERIMENTS

1 2 3 4

Analysis

Binding Assays

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

18

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

19

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLESGENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Only four guide (g) nucleotides (nt g8ndashg11) disordered

Structure of the Ago2-guide complex

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

20

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The guide 5prime end is anchored in the Ago2 MID (middle) domain and nucleotides g2ndashg7

are splayed in a helical conformation

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Nucleotides g14ndashg18 are threaded through a narrow channel formed between the PAZ and N domains of Ago2

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

21

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Consistent with the ability of Ago2 to bind guide RNAs of any nucleotide sequence the majority of contacts are made through hydrogen bonds and salt

linkages to the RNA sugar-phosphate backbone

7

Fig S2 Details of Ago2-guide interactions (A) Residues Y529 K533 Q545 K566 and K570 of the MID domain R812 of the PIWI domain and the carboxy-terminus of the protein make direct contacts to the guide 5-phosphate (B) Residues K566 of the MID domain and K709 R712 H753 Y790 S798 and Y804 of the PIWI domain make direct contacts to the phosphodiester backbone of nucleotides g3-g6 (C) Residues R277 R279 Y311 R315 and H316 of the PAZ domain make direct contacts to the 3 end of the guide RNA (D) Ago2 (colored) bound to a defined guide RNA (red) aligned to Ago2 bound to a mixture of cellular RNAs (grey protein pink RNA PDB ID 4OLA) Except for the appearance of guide nucleotides g12ndash20 and changes in a few loop residues the structures are nearly identical (RMSD of 0415 Aring for all equivalent Ca atoms) (E) Close up view of overlaid Ago2-guide structures in seed region colored as in (D)

Structure of the Ago2-guide complex

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

22

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Improvements in the electron density map allowed to observe amino acid residues 119 to 125 which fold into a hairpin loop that forms the end of the N-PAZ channel

and directs the guide 3prime end into the PAZ domain through contacts to g18

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2-guide complex

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

23

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Sequences of guide (red) and target RNAs (blue)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

(2-9 paired)

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Front and top views of Ago2 bound to guide and target RNAs

Structure of the Ago2 bound guide-target complex

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

24

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

The pocket appears to specifically recognize adenosine nucleotides because a target RNA with a t1-A bound Ago2 with almost threefold higher affinity than equivalent targets with U G or C t1 nucleotides

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

The t1- adenine inserts into a narrow pocket between the MID and L2 domains of Ago2 where

Ser561 hydrogen bonds to the adenine N6 amine

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

25

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

Aliphatic segments of residues R795 I756 and Q757 within the PIWI domain and I365 and T361 on helix-7 of the

L2 domain line the minor groove making extensive hydrophobic and van der Waals interactions with positions

2 to 7 of the guide-target duplex

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide-target complex

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

26

RESULTS

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

Binding experiments show that pairing to g8 can substantially contribute to the affinity of Ago2 for target RNAs

Structure of the Ago2 bound guide-target complex

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

27

RESULTS

An unrelated guide RNA displayed a smaller difference between the affinities for g2ndashg7 and g2ndashg8 complementary target RNAs revealing that the degree

to which pairing to g8 influences target affinity is dependent on the seed sequence

method generates relatively small amounts ofmaterial and is difficult to reproduce As an al-ternative we adapted a biochemical method for

purifying RISC loaded with a specified guidefrom cell lysates (20) to produce milligram quan-tities of recombinant Ago2 bound to a defined

21-nt guide RNA The ratio of Ago2 protein to guideRNA in the purified samples is 112 T 02 andprendashsteady-state kinetics showed that 990 T 86

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 609

Fig 2 Structure of Ago2 bound to seed-matched target RNAs (A) Sequences of guide(red) and target RNAs (blue) (B) Front and topviews of Ago2 bound to guide and target RNAs (C)Binding pocket for t1 adenine between L2 and MIDdomains (D) Equilibrium binding data for targetRNAs bearing different t1 nucleotides Mean valuesfromge three independent replicates T SEshown (E)Ago2 interrogates the guide-target minor grooveProtein is shown as a ribbon RNA in surfacerepresentation and interacting side-chains as stickswith dots Helix-7 (a7) is indicated with the arrow

Fig 3 Structuralanalysis of seed-pairing (A to C)Ago2-guide-targetcomplexes with pairingto (A) g2ndashg7 (B)g2ndashg8 or (C) g2ndashg9(D) Alignment ofg2ndashg9 structure(guide red targetblue) with g2ndashg7structure (guide pinktarget light blue)(E and F) Dissociationconstants of wild type(WT) and F811A Ago2proteins binding targetRNAs with variousdegrees of guidecomplementarityAgo2 was loaded withguide RNAs derivedfrom either (E) Sod1 or(F) miR122 Mean ofindependent tripli-cates TSEM

RESEARCH | RESEARCH ARTICLES

2

Materials and Methods Oligonucleotides Guide RNAs Sod1 5 p-rUrUrCrArCrArUrUrGrCrCrCrArArGrUrCrUrCrUrU 3 miR-122 5 p-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG 3 2OMethyl Capture oligos Sod1 5 Biotin-mUmCmUmUmCmCmCmAmCmGmAmCmUmUmCmAmUmAmAmAmUmGm UmGmAmAmAmCmCmUmU 3 miR-122 5 Biotin-mUmCmUmCmUmGmCmUmAmAmCmCmAmUmGmCmGmAmAmCmA mCmUmCmCmAmUmCmUmCmUmGmC 3 Competitor DNAs Sod1 5 AAGGTTTCACATTTATGAAGTCGTGGGAAGA 3 miR-122 5 GCAGAGATCAAGTGTTCGCATGGTTAGCAGAGA 3 Crystallized Target RNAs 5 rCrArArUrGrUrGrArArArA 3 5 rArArArUrGrUrGrArArArA 3 5 rArCrArUrGrUrGrArArArA 3 5 rCrCrArArArUrGrUrGrArArArA 3 Slicing Targets and Northern Blot Probes Sod1 5 rArArUrUrArArArArArGrArGrArCrUrUrGrGrGrCrArArUrGrUrGrArCrArCrCrUrUrArA 3 miR-122 5 CAAACACCATTGTCACACTCCA 3 Binding Assay Target RNAs Sod1 5 rUrCrCrCrUrUrArCrGrArCrGrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrCrArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArArArUrGrUrGrArArArA 3 5 rUrCrCrCrUrUrArCrGrArCrCrArCrArUrGrUrGrArArArA 3 miR-122 5 rArCrCrUrGrCrArGrUrCrGrUrUrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrCrArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArArCrArCrUrCrCrArArA 3 5 rArCrCrUrGrCrArGrUrCrGrUrCrArUrCrArCrUrCrCrArArA 3

Structure of the Ago2 bound guide-target complex

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

28

RESULTS

GENE REGULATION

Structural basis formicroRNA targetingNicole T Schirle Jessica Sheu-Gruttadauria Ian J MacRae

MicroRNAs (miRNAs) control expression of thousands of genes in plants andanimals miRNAs function by guiding Argonaute proteins to complementary sites inmessenger RNAs (mRNAs) targeted for repression We determined crystal structuresof human Argonaute-2 (Ago2) bound to a defined guide RNA with and without targetRNAs representing miRNA recognition sites These structures suggest a stepwisemechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initialtarget pairing Pairing to nt 2 to 5 promotes conformational changes that exposent 2 to 8 and 13 to 16 for further target recognition Interactions with the guide-targetminor groove allow Ago2 to interrogate target RNAs in a sequence-independentmanner whereas an adenosine binding-pocket opposite guide nt 1 further facilitatestarget recognition Spurious slicing of miRNA targets is avoided through an inhibitorycoordination of one catalytic magnesium ion These results explain the conservednucleotide-pairing patterns in animal miRNA target sites first observed over twodecades ago

MicroRNAs (miRNAs) are small [~22 nu-cleotides (nt)] RNAs with regulatory rolesin plants and animals miRNAs functionwithin RNA-induced silencing complexes(RISCs) which contain a member of the

Argonaute protein family (1) Argonaute uses themiRNA as a guide for identifying complementary

target mRNAs which then leads to silencing ofthe targeted messages via translational repres-sion and degradation (2) More than 1000miRNAsare encoded in the human genome and over 50of mammalian protein-coding genes contain aconserved miRNA target site (3) ConsequentlymiRNAs contribute to diverse physiological pro-

cesses in mammals including epithelial regen-eration (4) cardiac function (5) ovulation (6) andthe progression of cancer (7)Perfect complementarity between miRNAs

and their targets is not necessary for silencingand some miRNA nucleotides are more im-portant than others (8 9) Specifically pairingto the miRNA ldquoseed regionrdquo (nt 2 to 7 or 2 to8 from the 5prime end) is the most evolutionarilyconserved feature of miRNA targets in animals(10ndash14) Crystal structures of human Argonauteproteins show nt 2 to 6 of the guide RNA boundin a prearranged A-form conformation whichwas proposed to minimize the entropic costassociated with forming a stable duplex withtarget RNAs (15ndash17) However nucleotides out-side of the seed region in these structures weremostly disordered and no structure of any eu-karyotic Argonaute bound to a target RNA hasbeen reported

Guide RNAs are threaded through theN-PAZ channel

Structural insights into eukaryotic Argonauteproteins have been thwarted by the challengeof separating Argonaute from copurifying cellu-lar RNAs (15 18) Although a protocol for purify-ing RNA-free Ago2 has been reported (19) the

608 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Department of Integrative Structural and Computational BiologyThe Scripps Research Institute La Jolla CA 92037 USACorresponding author E-mail macraescrippsedu

Fig 1 Structure of the Ago2-guide complex (A) Schematic of the Ago2 primary sequence Front and top views of human Ago2 bound to a definedguide RNA (red) Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2) (B) GuideRNA omit map contoured at 2s (blue mesh) (C) Nucleotides g2ndashg5 are exposed whereas Ago2 occludes nucleotides g6 and g7 (D) The 3prime half of theguide is threaded through the N-PAZ channel (E) View down the N-PAZ channel

RESEARCH | RESEARCH ARTICLES

In moving from the guide- only to the target-bound conformation helix-7 shifts ~4 Aring to interact with the minor groove of the guide-target duplex The

movement of helix-7 is required to avoid steric clashes with target nt t6 and t7 and is therefore necessary for target pairing beyond g5

groove of the guide-target duplex Aliphatic seg-ments of residues R795 I756 and Q757 withinthe PIWI domain and I365 and T361 on helix-7of the L2 domain line the minor groove makingextensive hydrophobic and van der Waals in-teractions with positions 2 to 7 of the guide-target duplex (Fig 2D) These minor groovecontactsmay explainwhyGUwobble base pairsin which the guanine unpaired exocyclic aminoalters minor groove shape and electrostatic po-tential (23) reduce Argonaute target affinity andare not commonly observed in miRNA targetsites (13 24 25) (Single-letter abbreviationsfor the amino acid residues are as follows AAla C Cys D Asp E Glu F Phe G Gly H HisI Ile K Lys L Leu M Met N Asn P Pro QGln R Arg S Ser T Thr V Val W Trp and YTyr In the mutants other amino acids weresubstituted at certain locations for exampleF811A indicates that phenylalanine at position811 was replaced by alanine)In contrast to positions 2 to 7 Ago2 does not

contact the minor groove at positions 8 and 9

suggesting that the protein is more tolerant ofmismatches in this region (Fig 3 A to C) In-deed the guide-target duplex with g8ndashg9 mis-matches has distortions away from A-form dueto staggering of the mismatched bases (Fig 3A)The distortions are limited to positions 8 and 9indicating that pairing status at g8 and g9 doesnot perturb guide-target duplex structure inpositions 2 to 7 (Fig 3D) However binding ex-periments show that pairing to g8 can substan-tially contribute to the affinity of Ago2 for targetRNAs (Fig 3E and fig S8) An unrelated guideRNA displayed a smaller difference betweenthe affinities for g2ndashg7 and g2ndashg8 complemen-tary target RNAs revealing that the degree towhich pairing to g8 influences target affinityis dependent on the seed sequence (Fig 3F)

Narrowing of the central cleft restrictspairing past g8

Although complementarity to g8 increased theaffinity of Ago2 for target RNAs extending com-plementarity to g9 and g10 did not enhance

affinity further (Fig 3 E and F) In fact bothsequences displayed a modest decrease in affi-nity for targets complementary to g2ndashg9 com-pared with g2ndashg8 indicating that pairing to g9is actually detrimental to stability of the Ago2-guide-target complex t9 stacks against F811 inthe 2 to 9 paired structure (Fig 4A) Howeveran F811A mutation did not markedly alter theaffinity for full-length target RNAs (Fig 3 E andF) Moreover the complex lacks sufficient spacenecessary to accommodate target nucleotidesbeyond t9 (Fig 4 B and C) and the t9 nucleotidewas disordered in crystals containing a longertarget RNA which included mismatches to g11(Fig 4 D and E) We conclude that the observedconformation of Ago2 can only accommodatepairing to g9 when using short targets that endat t9 (such as those used to facilitate crystalli-zation) and that pairing to g9 on longer targets(such as those in the 3prime UTR of a mRNA) requiresfurther opening of the Ago2 central cleft Wesuggest that opening the cleft involves confor-mational changes responsible for the decrease

SCIENCE sciencemagorg 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 611

Fig 5 Comparison of Ago2-guide and Ago2-guide-target structures (A) Helix-7 (a7) shifts toaccommodate pairing to target RNAs The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored) (B) The PAZ domain andhelix-7 move as a rigid body Superposition of pro-tein components from Ago2-guide (semitransparent)and Ago2-guide-target (opaque) structures Arrowsindicate movement from guide-only to guide-targetstructures Dashed line marks hinge in the L1L2domains (C and D) Contacts to the guide RNAsupplemental region in the (C) guide-only and(D) target-bound structures (E) The supplementalregion (g13ndashg16) adopts an exposed helical con-formation in the Ago2-guide-target structure (F) Il-lustrated model for seed plus supplemental pairing

RESEARCH | RESEARCH ARTICLES

Structure of the Ago2 bound guide RNA with and without target RNAs

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

29

RESULTS

Magnesium ion (green) is bound to the D597 carboxylate side chain the V598 main chain carbonyl and four water molecules (brown spheres)

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

in target affinity associated with pairing to g9This model may explain why complementaritybeyond g8 is not a well-conserved feature ofvertebrate miRNA targets sites (11)

A structural model for miRNA targeting

Structures of human Ago1 and Ago2 and yeastAgo1 indicate that in the absence of target RNAeukaryotic Argonaute proteins kink their guideRNAs at the end of the seed region (15ndash18) Thekink appears to be stabilized by helix-7 whichinserts a hydrophobic residue (I365 in Ago2)between g6 and g7 In moving from the guide-only to the target-bound conformation helix-7shifts ~4 Aring to interact with the minor groove ofthe guide-target duplex (Fig 5A) The movementof helix-7 is required to avoid steric clasheswith target nt t6 and t7 and is therefore nec-essary for target pairing beyond g5 (fig S9)Movement of helix-7 also releases the con-straints on the guide RNA relaxing the kink andallowing g6 and g7 to adopt an A-form confor-mation for target pairing Thus the guide andprotein act synergistically to recognize targetRNAs movement of helix-7 to accommodatetargets beyond g5 also releases g6 and g7 fa-cilitating the additional base pairing Forma-tion of these base pairs generates a guide-target

duplex with a minor groove that provides anew binding surface for helix-7 stabilizing theopened conformation Conversely mismatchesor GU wobble pairs would present a distortedminor groove to helix-7 which would then bemore likely to shift back toward the guide RNAand displace the mispaired target

Seed pairing opens the N-PAZ channelfor supplemental pairing

Superimposing guide-only and guide-target struc-tures of Ago2 indicates that helix-7 and the PAZdomain move as a discrete rigid body relative tothe MID PIWI and N domains (Fig 5B) Thehinge for this conformational change residesin the base of helix-7 and extends across theb-sheet in the L1 domain We suggest that themovements in helix-7 induced by seed pairingare propagated to positional shifts in the PAZdomain leading to a widening of the N-PAZchannel Mutation of F181 which resides in thehinge inhibits small RNA duplex unwinding dur-ing RISC-loading (26) suggesting that relatedconformational changes are involved in passen-ger strand removalWidening of the N-PAZ channel is accompa-

nied by repositioning of the 3prime half of the guideRNA with g11ndashg16 shifting to adopt a near A-

form conformation (Fig 5 C and D) Helicalstacking is disrupted after g16 by P67 of the Ndomain and electron density for g17ndashg19 isweak which is indicative of conformational het-erogeneity However density for g20 and g21is visible with the 3prime end of the guide bound tothe PAZ domain The Watson-Crick faces of g13ndashg16 which can supplement repression of targetsites with weak seed pairing (27) are splayed outtoward the solvent in a manner reminiscent ofthe seed region in guide-only structures (Fig 5D)We suggest that seed pairing is coupled to a re-arrangement in the 3prime half of the guide thatfacilitates target interactions in the supplemen-tal region Nucleotides g12 and g13 are partiallyoccluded by a PIWI domain loop (residues 602to 608) indicating that target pairing to thesupplemental region may nucleate at g14ndashg16and extend back into the central cleft We sug-gest that target RNAs paired to the seed mayexit the central cleft and extend over the middleof the PIWI domain to pair with the supple-mental region on the other side (Fig 5 E and F)This would allow the complex to maximize guide-target interactions while avoiding the topolog-ical issues and entropic costs associated withwrapping the target and guide RNAs aroundeach other within the central cleft (3) Consistent

612 31 OCTOBER 2014 bull VOL 346 ISSUE 6209 sciencemagorg SCIENCE

Fig 6 Inactive magnesium ion in the Ago2 active site (A and B) Magne-sium ion (green) is bound to the D597 carboxylate side chain the V598main chain carbonyl and four water molecules (brown spheres) 2Fo-Fcmap (blue mesh) was contoured at 15s (A) and Fo-Fc magnesium omitmap (green mesh) was contoured at 15s (B) (C) Active site of Ago2(gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH)

(D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR)Metals ions are shown as spheres (E) Ago2 active site aligned with Bacillushalodurans RNase H (pink with red magnesium ion PDB ID 2G8H) (F) Align-ment of Ago2 (gray with green magnesium) TtAgo (blue) and B haloduransRNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the activeposition

RESEARCH | RESEARCH ARTICLES

Alignment of Ago2 (gray with green magnesium) TtAgo (blue) and B halodurans RNase H (pink) shows the Ago2 magnesium shifted 15 Aring from the active

position

Structure of inactivated magnesium ion in the slicer active site

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

30

CONCLUSION

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

CONCLUSION

31

Stepwise mechanism in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing Pairing to nt 2 to 5 promotes conformational changes

that expose nt 2 to 8 and 13 to 16 for further target recognition

Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner whereas an adenosine binding-pocket

opposite guide nt 1 further facilitates target recognition

Slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion by bound to the D597 carboxylate side chain the V598

main chain carbonyl and four water molecules

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

32

REFERENCES

(1) Ha M and Kim V N (2014) Regulation of microRNA biogenesis Nat Rev Mol Cell Biol 15 509-524 (2) Hutvagner G and Simard M J (2008) Argonaute proteins key players in RNA silencing Nat Rev Mol Cell

Biol 9 22-32 (3) Jinek M and Doudna J A (2009) A three-dimensional view of the molecular machinery of RNA

interference Nature 457 405-412

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015

Schirle N T et al (2014)

Presented by Bundit Boonyarit 5814400587

DeptBiochemistry FacScience Kasertsart University

tructure basis for microRNA targetingS

December 27 2015