1 rna interference mbg-487. 2 'rna interference' scoops nobel prize for medicine in 2006...

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RNA interference

MBG-487

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'RNA interference' scoops Nobel prize for Medicine in

2006• Two US scientists won the Nobel prize for

Medicine on Monday for their discovery of RNA interference pioneering work that has revolutionised the field of molecular biology and is leading to powerful genetic medicines.

• Andrew Fire at Stanford University in California, and Craig Mello at the University of Massachusetts in Worcester.

• The pair "discovered a fundamental mechanism for controlling the flow of genetic information”

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Gene silencing

• In 1997, the Fire-Mello team discovered that they were able to “silence” certain genes in a cell's DNA by using two strands of RNA molecules. This prevented the genes from being expressed. Dubbed RNA interference (RNAi), the finding has become an extremely useful research tool, because it allows genetic researchers to “knock-out” specific genes, observe the consequent disruptions, and so determine exactly what the gene does.

• RNA interference is crucial for the regulation of gene expression. It participates in the defense against viral infections, and keeps jumping genes in check. RNA interference is currently used extensively in basic science as a method to study gene function - this research should lead to new future therapies.

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• It is being used as a genome-wide tool, to search thousands of genes in a cell and screen for their function. The technique has already revealed genes responsible for muscle problems and diabetes.

• A byproduct of the discovery of RNAi was the finding that although cells in the human body only contain one strand of RNA, they do have “micro-RNA” tiny sections of RNA that can act a little like double-stranded RNA and also silence the activity of certain genes. Experts believe the body uses micro-RNA in its immune response.

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What We’ll Cover

• What is RNAi/ useful terms

• Brief history of RNAi

• Biogenesis and mechanisms of action

• Applying RNAi to model systems

• Endogenous RNAi: miRNA in the genome

• New frontiers for RNA

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What is RNA Interference (RNAi)

• “The Process by which dsRNA silences gene expression...”

• Generally: Post transcriptional level (PTGS)

• Degradation or translation inhibition

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Handy RNAi Terms

• dsRNA: double stranded RNA, longer than 30 nt

• miRNA: microRNA, 21-25 nt. – Encoded by endogenous genes. – Hairpin precursors– Recognize multiple targets.

• siRNA: short-interfering RNA, 21-25 nt.– Mostly exogenous origin.– dsRNA precursors– May be target specific

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An Arbitrary Distinction?• miRNA vs. siRNA?• Discovered in different ways• Similar biogenesis• Share common pathway components• Common pathway outcomes• Understanding of miRNA comes from

research on siRNA and vice versa• Some use terms interchangeably

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A Brief History of RNAi

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An Unexpected Result• Late 1980s, Rich Jorgenson and group• More than a decade ago, a team led

by molecular biologist Rich Jorgensen, was trying to deepen the flowers' hues. Jorgensen's strategy hinged on boosting the activity of the gene for chalcone synthase, an enzyme involved in production of the anthocyanin pigments. The researchers hooked up the gene to a powerful promoter sequence and ferried this genetic construct into their petunias. However, instead of deep purple, many of the flowers grew up variegated, or virgin white

• chalcone synthase for deeper purple petunias

• Got white and variegated• Co-suppression: both endogenous and

introduced genes silenced [Napoli et al., 1990]

• post-transcriptional gene silencing - PTGS – but what is the causative factor?

From: Gura,2000.

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in the C. elegans...

• Antisense RNA injection method for gene inactivation

• 1995: characterization of Par1 by Sue Guo– Essential for embryo polarity

• Did antisense Par1 RNA injections– Results in embryonic lethality

• Sense Par1 RNA injections gave same result!

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Some Sharp Reasoning

• Fire and Mello, 1998• Both sense and antisense RNAs sufficient for

silencing• Silencing can persist, even though RNA is easily

degraded• RNA for silencing often generated using

bacteriophage RNA polymerases– Specific, but can also make ectopic transcripts– Maybe some dsRNA in these preparations?

• Could dsRNA be mediating a new silencing mechanism?

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Their Experiment• C. elegans Unc-22 inactivation

– Null phenotype = uncoordinated twitching

• Injected sense, antisense, or both into c. elegans gut• dsRNA was orders of magnitude more effective than

ssRNA– Effective even in tiny amounts

• Unc-22 null phenotype also seen in progeny of injected worms

• Inactivation was due to degradation of target mRNA• Coined the term “RNA interference” [Fire et al., 1998]

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siRNA Identified• 25bp species of dsRNA found in plants with

co-suppression [Hamilton and Baulcombe, 1999]– Not in other plants– Sequence similar to gene being suppressed

• Drosophila: long dsRNA “triggers” processed into 21-25bp fragments [Elbashir et al., 2001]– Fragments = short interfering RNA (siRNA)– siRNA necessary for degradation of target

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Meanwhile, Back in C. elegans…

• Discovery of the first miRNA, lin-4– Non-coding, 22nt RNA

• Identified in screen for defects in timing of larval development– lin-4 mutation – ectopic larval stage 1-like cell divisions at

later stages– lin-14 mutations – reciprocal phenotype, same regulatory

pathway as lin-4– lin-4 negatively regulates lin-14 translation

• lin-4 partially complementary to conserved sites in lin-14 3’UTR [Lee et al., 1993]– Required for negative regulation of lin-14– lin-4 binds these sites

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• Victor Ambros' laboratory were working on how the timing of development is controlled in Caenorhabditis elegans, when they discovered a small temporal RNA (stRNA) that was crucial to this process, called lin4.

• The expression of lin4 RNA during the first larval stage seemed to trigger the downregulation of mRNAs that specified the temporal progression of cell fates during development.

Meanwhile, Back in C. elegans…

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Unique Occurrence?• No other miRNAs found for 7 years!• Second miRNA – let-7 [Reinhart et al., 2000]

– Non coding, 21nt RNA– Regulates lin-14 in same way as lin-4

• Maybe miRNA is in other organisms?• Homologues of lin-4 escaped bioinformatics• Let-7 Homologs were easily detected

[Pasquinelli et al., 2000]– Drosophila, sea urchins, mice, humans...– Indicates RNAi general conserved mechanism

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An Ancient Process

• Predates evolutionary divergence of plants and worms [Novina and Sharp, 2004]

• Silencing of viruses and genetic elements

• NOW...– miRNA and siRNA – same mechanism– Increasingly detailed knowledge

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The short story of gene silencing - the main characteristics of short RNAs.

Novina and Sharp, 2004

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Biogenesis and Mechanism of RNAi

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RNAi: Two Phase Process

• Initiation– Generation of mature siRNA or miRNA

• Execution– Silencing of target gene– Degradation or inhibition of translation

23He and Hannon, 2004

Initiation

Execution

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The RNA Components

• siRNA and miRNA

• How do they arise?

• What are their characteristics?

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miRNA Biogenesis• Transcribed from endogenous gene as pri-miRNA

– Primary miRNA: long with multiple hairpins– Imperfect internal sequence complementarity

• Cleaved by Drosha into pre-miRNA– Precursor miRNA: ~70nt imperfect hairpins– Exported from nucleus

• Cleaved by Dicer into mature miRNA– 21-25nt– Symmetric 2nt 3’ overhangs, 5’ phosphate groups

Novina and Sharp, 2004

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miRNA Biogenesis miRNAs are encoded in the genomes of all multicellular organisms

studied so far. They can be expressed either from an intergenic cluster, or from single genetic regions.

A mature miRNA is then assembled into a ribonucleoprotein (miRNP) complex.This complex most commonly binds to the 3'-untranslated sequences of particular mRNAs through partially complementary sequences, and prevents the mRNAs from being translated into protein.

Base-pairing with the 7-8 nucleotides near the 5' terminus of the miRNA is essential here. Numerous miRNP complexes bind to the 3'-untranslated sequences of mRNAs and cooperate for effective translational silencing through interactions (green arrow) that are still unclear.

In a few cases, the miRNA is exactly or nearly exactly complementary to a site in an mRNA, and this results in cleavage and degradation similar to that observed with siRNAs.

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siRNA Biogenesis• Dicer cleaves long dsRNA into siRNA 21-25nt

– dsRNA from exogenous sources, a viral intruder or a rouge genetic element

– Symmetric 2nt 3’ overhangs, 5’ phosphate groups• Evidence for amplification in C. elegans and plants

– Allows persistence of RNAi?

Novina and Sharp, 2004

Strand blue is degraded

Strand yellow is used

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In worms and plants, the antisense strand of the siRNA might first be used in an amplification process. The antisense strand, bound by an RNA-dependent RNA polymerase (RdRP) enzyme, can pair up with a complementary mRNA (green) and act as a start point for the synthesis of a new long dsRNA. Dicer is then required to generate new siRNAs (red), which are specific to different sequences on the same mRNA. Again, the target mRNA is destroyed.

siRNA Biogenesis

Novina and Sharp, 2004

Strand blue is degraded

Strand yellow is used

Target complementary mRNA

Target complementary mRNA

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The Protein Components

• What are they?

• How do they function?

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Endogeneous siRNAs

• microRNA (miRNA) blocks mRNA translation through

imperfect complementary binding to miRNA recognition

elements (MREs) within the 3’UTR of the targets.

• transcribed in nucleus as a pri-miRNA then processed by

Drosha (RNase III enzyme) to a hairpin RNA of ~ 70 nts (pre-

RNA);

• exported into the cytoplasm by exportin-5.

• pre-miRNA is cleaved by Dicer into a double stranded 21-23

nucleotide RNA duplex incorporated into a RISC like complex.

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Drosha

• RNase III enzyme, contains two canonical RNAse III domains.

• Found in nucleus and is concentrated in nucleolus.

• Drosha knockdown- accumulation of 12S and 32S precursor rRNAs.

• Drosha cleaves pri-miRNAs into pre-miRNAs.

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A model for microRNA biogenesis, trafficking and assembly into RISC. (a) miRNAs are transcribed in the nucleus and pri-miRNAs are processed by Drosha into miRNA precursors, which have the two-nucleotide 3′ overhang characteristic of RNaseIII cleavage. (b) The two-nucleotide 3′ overhang end structure of the miRNA precursor is recognized by Exportin-5, a Ran-GTP-dependent nuclear export factor. The miRNA is transported into the cytoplasm. (c) The miRNA precursor is cleaved by Dicer, which probably uses the PAZ domain to specifically recognize and bind the two-nucleotide 3′ overhang. Dicer cleavage of the miRNA precursor liberates a 22 nt mature miRNA. Having been processed by two RNaseIII enzymes, the miRNA now has symmetrical end structures.

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Drosha

• Pre-miRNAs (~70nt) produced by Drosha have the two nt -3’ overhang end structure left by the staggered cut of RNAse enzymes.

• Exportin-5 mediation may need these overhangs.

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Drosha• Processes pri-miRNA into pre-miRNA

– Leaves 3’ overhangs on pre-miRNA

• Nuclear RNAse-III enzyme [Lee at al., 2003]– Tandem RNAse-III domains

• How does it identify pri-miRNA?– Hairpin terminal loop size– Stem structure– Hairpin flanking sequences

• Not yet found in plants– Maybe Dicer does its job?

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Dicer• Rnase III type enzyme.• ATP-dependent • Dicer translocates along its dsRNA target.• Cuts the dsRNA into pieces or pre-miRNA

– Leaves 3’ overhangs and 5’ phosphate groups

• Efficiency of Dicer is proportional to the length of the target– the longer the dsRNA, the greater the amount of siRNA produced and

increased efficiency of silencing.

• Functional domains in Dicer [Bernstein et al., 2001]– Putative helicase– PAZ domain– Tandem RNAse-III domains– dsRNA binding domain

• Dicer-mediated cleavage of dsRNA starts at the termini of the dsRNA, continues with excision of small dsRNA fragments of defined length.

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siRNA

55’’

33’’ 55’’

33’’

3` hydroxyl5`phosphate groups3` overhang of two unpaired nucleotides on each strand.

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Why 3′ overhangs?

• Possible functions Enable siRNA unwinding Promote efficient RISC assembly.

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• Interacts with Rde-4 and Rde 1 (C.elegans).Detect, retain and present dsRNA to Dicer

• ATP dependent translocation of the enzyme along its dsRNA target.

• Initiates cleavage at the ends of dsRNA.

• Size limitation.

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RISC (RNA Induced Silencing Complex)

• A riboprotein complex.

• Has nuclease activity

• It occurs as inactive (RISC) and as an active (RISC*).

• Following unwinding of siRNA duplexes, RISC is converted to RISC* by an ATP-dependent step.

• RISC* is associated with only one strand of the siRNA. These suggest that RISC may have an ATP-dependent helicase activity or a helicase associated with it.

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RISC (RNA induced silencing complex)

• 2 RNA binding 2 RNA binding proteinsproteins

• RNA/DNA RNA/DNA HelicaseHelicase

• Translation Translation Initiation FactorInitiation Factor

• Transmembrane Transmembrane ProteinProtein

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RISC

• RDE-4/R2D2 (RNA-binding) facilitate transport to RISC.

• RISC=ribonucleoprotein complex, co-purifies with AGO2 protein from S2 cells.– Argonaute family proteins (gives specificity since they are in

multiples in different species).

• They have a PAZ domain

• Have PIWI domain, inhibits Rnase III/dsRNA binding domain of

DICER (miRNA/siRNA release?)

• Other proteins associate with Argonaute complexes.

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RISC

• Functions in:– mRNA cleavage– Translational suppression.

• RISC may bind to polyribosome and stall translation.

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A 22 nt miRNA or siRNA is recognized by the PAZ domain of an Ago protein, and incorporated into RISC; R2D2/RDE-4 facilitates transfer of miRNAs or siRNAs into RISC. RISC components identified in the Drosophila S2 cell system include, besides Ago, TSN-1, VIG and dFRX. An additional complex has been described in mammalian cells, which contains miRNAs, Ago2 and Gemin3 and Gemin4. An early step in RISC maturation is the unwinding of the miRNA duplex into a single-stranded form. Depending upon its specific components, RISC may target homologous mRNA for cleavage, stall mRNA translation, perhaps in complex with polyribosomes, or induce chromatin modification and transcriptional gene silencing (this activity has only been directly observed in S. pombe)

RISC ASSEMBLY

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RNA Induced Silencing Complex (RISC)

• RNAi effector complex– Critical for target mRNA degredation or translation inhibition

• Not well characterized: 4 subunits? More?• Activities associated with RISC

– Helicase– Endonuclease and exonuclease “Slicer” (or is it Dicer?)– “homology seeking”/RNA binding

• Preferentially incorporates one strand of unwound RNA [Khvorova et al., 2003]– Antisense– How does it know which is which?

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RISC Preference for Antisense RNA

• Helps ensure specificity for target

• 5’ stability of siRNA and miRNA duplex strands often

different

• The strand with less 5’ stability usually incorporated

into RISC [Schwarz et al., 2003]

– Due to easier unwinding from one end?

• If strand stability is similar (rare), strands

incorporated at similar frequency [He and Hannon,

2004]

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Argonaute (Ago)• Consistently co-purifies with RISC [Hammond et al.,

2001]• “Homology seeking” activity?

– Binds siRNA and miRNA [Ekwall, 2004]– Distinguishes antisense strand [Novina and Sharp, 2004]

• Multiple Ago family proteins– Different RISCs?– Tissue specific? Developmentally regulated?

• Evidence for different RISCs [Tijsterman et al., 2004]– Drosophila Dicer1 vs Dicer2/R2D2– Inhibition vs. degradation [Lee et al., 2004]

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RNA Dependent RNA Polymerase (RdRP)

• RdRP activity found in plants and C. elegans• May explain efficiency of RNAi• Required for RNAi?

– Not found in mammals or drosophila– RdRP deficient plants and worms... Results not decisive

• Random degenerative PCR [Lipardi et al., 2001]– Proposed mechanism– siRNA acts as primer for elongation on target mRNA– Result: more long dsRNA

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Amplification of RNAi effect

• In non-mammals (fungi, plants, and invertebrates), the unwound siRNA is amplified.

• acts as a primer for an RNA-dependent RNA polymerase (RdRP), which uses the target mRNA as a template to produce new dsRNA.

• The new siRNA gets cleaved by Dicer and amplification of the original RNAi trigger takes place.

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Getting the Job Done

• Translational inhibition

• Transcript degradation

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Translational Inhibition

• Imperfect match between siRNA or miRNA in RISC and target mRNA

• RISC usually binds 3’ UTR

• Mechanism of inhibition... ????

He and Hannon, 2004

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mRNA Degradation

• Perfect complementarity between siRNA or miRNA in RISC and the target mRNA

• Cleavage by RISC Slicer activity– Could be Dicer?– Other endo/exonucleases?– Recruitment of other

components?Novina and Sharp, 2004c

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Other Effects?• RNAi process also work on transcriptional level?

[Volpe et al., 2002]– Plants, C. elegans, Drosophilia– Via chromatin modification [Mochizuki et al., 2002]

• Heterchromatin formation machinery fairly well characterized

• What’s the connection?– Argonaute– RITS complex – RNA-induced initiation of transcriptional

silencing [Verdel et al., 2004]– RITS could mediate targeted heterchromatin formation

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RITS Connects RNAi and Heterchromatin Formation Machinery

Novina and Sharp, Nature 430: 2004

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shRNA and siRNA Mediated Gene Silencing. Different delivery strategies and the processing of shRNAs in the cell are shown.

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Applying RNAi to Model Systems

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Why?

• Quick way to do loss-of-function studies– Targeting takes long time, lots of work– Not all loci amenable to targeting– Cheap

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RNAi in plants, C. elegans, Drosophila

• Introduction of dsRNA sufficient for RNAi– In vitro transcription– Chemical synthesis

• Remarkably straightforward: C. elegans– Feed E.coli expressing dsRNA [Timmons and Fire,

1998]– Soak them in dsRNA [Tabara et al., 1998]

• Common methods: transfection or microinjection of dsRNA– Effect lasts days– Passed onto daughter cells/progeny

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Great Potential

• Whole genome RNAi screening– What do all the proteins do?– Knock each down!

• Done in C. elegans– 19 757 protein coding genes (predicted)– 16 757 inactivated using RNAi

• Ravi Kamath et al., 2003– New standard for systematic genome wide

functional studies

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Generation of Bacterial Feeding Library

• C. elegans primer set from Research Genetics– 19 213 primer pairs; each for protein coding gene

• Generated PCR products• Cloned into dual promoter vector

– Both sense and antisense strands transcribed under induction conditions

• Result: 16 757 bacterial strains– 86.3% of predicted genes– Remaining: PCR failures, cloning failures

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Induction of RNAiTuschl, 2003

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Assayed Phenotypes: Examples

• Emb – embryonic lethal• Ste – sterile• Gro – slow growth• Adl – adult lethal• Lvl – larval lethality• Lva – larval arrest• Bmd – body

morphological defects

• Unc – uncoordinated• Clr – clear• Prz – paralyzed• Lon – long• Mlt – moulting defects• Egl – egg laying defects• Him – high incidence of

males

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Results• 10% of targeted genes gave obvious

phenotypes• Highly conserved genes most likely to give

aberrant phenotype– More likely to be essential– DNA synthesis, cell cycle control

• “New” genes unlikely to have detectable phenotype– Lots of gene duplications– Very specialized or redundant functions

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Results

• Examined domains in proteins knocked down• Non-lethal phenotypes from “more recent” domains

– Animal specific domains– Example: Immunoglobulin-like repeats

• Non-viable phenotypes from inactivation of proteins with “ancient” domains– Domains shared with plants and lower eukaryotes– Domains needed for survival evolutionarily preserved

• Genomic clustering of genes yielding phenotypes– Common origins or regulatory mechanisms?

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One Step Further...• Ashrafi et al. (2003) used same RNAi library• Screened for particular phenotypic readout

using a cellular marker– Interested in fat storage regulation

• Found 417 genes involved in fat storage– Many conserved – new obesity drug targets?

[Tuschl, 2003]

• Repeated RNAi with mutant lines– Known defects in fat storage– Allowed new genes to be placed in fat regulation

pathways

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How About Mammals?

• Application of RNAi to mammalian system promising for functional studies

• Evidence of RNAi in mammals was harder to establish

• Methods for RNAi not a straightforward

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Interferon response• More than 100 genes can be activated by IFNs and some

encode enzyme that are dsRNA-binding proteins.

• PKR (protein kinase R), which phosphorylates and inactivates eukaryotic initiation factor-2alpha, inhibits mRNA translation - IFN response

• Synthetic 21-22 nt siRNA can bypass the initial Dicer step, and do not induce IFN response.– Removal of the 5’ triphosphate of the transcripts prevents IFN

response. Commercially available siRNAs have free hydroxyl groups at the 5’ ends, which are phosphorylated intracellularly.

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Non-Specific Silencing via Antiviral Pathway

McManus and Sharp, 2002

dsRNA-dependent protein kinase(antiviral detector protein)

PKR also activates RNAsel enzyme (component of the antiviral pathway)

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Getting Around the Problem

• Critical observation [Elbashir et al., 2001]– Size matters– siRNA (21-22nt) mediate mammalian RNAi– Introducing siRNA instead of dsRNA prevents non-

specific effects

• Application via transient transfection– Don’t see persistent or propagative effect as in C.

elegans etc.– No RdRP (RNA dependent RNA Polymerase)

activity identified– Chemically synthesized – In vitro transcription

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Empirical siRNA Design Rules• 21nt long, with 2nt 3’ overhangs• Avoid introns and UTRs• Avoid regions >50% GC content• Use stringent BLAST to help ensure

specificity

• Limitations:– Inability to interact with RISC– Target inaccessibility (structural constraints?)– Instability of the siRNA

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Still Not Too Efficient

• Usually need to design several siRNAs to get an effective one

• Could use a mixture of siRNAs– Recombinant Dicer available– Use in vitro to cleave dsRNA

• Problems:– Increased possibility of non-specific targeting– Low effective siRNA concentration– Don’t know which siRNA is most potent

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Rational Design of siRNA

• Arising from research on RISC assembly• RISC contains one strand of the siRNA duplex

[Martinez et al., 2002]• Needs to be the antisense strand to find right target• Can we direct preferential incorporation of the

antisense strand into RISC?• Observation: 5’ end of an siRNA strand is

incorporated into RISC most efficiently [Schwarz et al., 2003]

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Rational Design Points

Mittal, 2004

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Stable RNAi in Mammals• Vector driven methods• Expression of sense and antisense siRNA

– Stable production of siRNA with 3’ overhangs

• Expression of pre-miRNA like RNAs– RNA that folds into hairpin loops with 3’ overhangs– Act like pre-miRNA dicer substrates

• Some evidence for induction of interferon response? [Bridge et al., 2003; Sledz et al., 2003]

• Could do inducible, time, and tissue specific RNAi– Therapeutic potential– Effective delivery an issue...

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Efficiency of siRNA

• RISC demonstrates strand bias that is related to the relative strength of the base pairing at each end. – siRNA and miRNA duplexes should have a

weaker A-U base pairs at the 5’-end and stronger G-C base pairs at the 3’-end of the antisense strand.

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siRNA selectivity

• Carefully screened (BLASTED) siRNA sequences needed. Blast your sequence against the nucleotide and EST databases, discard any with greater than 17 consecutive bp match.

• At least two siRNAs per gene of interest with additional scrambled and mismatched controls.

• Off-target effects: knock-down of unrelated targets.– Induction of miRNA related pathways– siRNA stimulation of a subset of genes involved in interferon

response.

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siRNA design at ambion.com

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siRNA design at ambion.com

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siRNA design at ambion.com

T7 promoter seq.

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siRNA and shRNA design

• 50-60% of siRNAs designed according to

the publicly available design criteria are

functional (reduction of target mRNA at a

level of 70% after 48 hours).

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More on selection criteria

• Specific base compositions at key positions along the 19

core siRNA base pairs

• Thermodynamic base-pairing profiles defining ‘regional

base compositions’ (GC content)

• Base composition of 3’ overhangs

• Positions along the targeted mRNA

• Lack of variability of the targeted mRNA (lack of SNPs).

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More on selection criteria

• First 75-100 nucleotides of any mRNA

should be avoided since they may contain

protein-binding regulatory sequences

(5’UTR).

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Always design negative controls by scrambling targeted siRNA sequence. The control RNA should have the same length and nucleotide composition as the siRNA but have at least 4-5 bases mismatched to the siRNA. Make sure the scrambling will not create new homology to other genes.  

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More at ambion.com

• http://www.ambion.com/techlib/tn/113/14.html

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RNAi in vivo

• Exogenous reporter systems:– Co-injection of a luciferase-encoding plasmid

and anti-luciferase siRNA in mice resulted in lower hepatic luciferase expression relative to control siRNA treatment [McCaffrey et al., 2002; RNA interference in adult mice. Nature 418:38-39].

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Exogenous reporters

• light emitted from mice co-

transfected with the luciferase

plasmid pGL3-control and

either no siRNA, luciferase

siRNA or unrelated siRNA (A).

Delivery: coinjection into the

liver; mice given 3 mg luciferin

and imaged.

• Luciferase inhibition by an

average of 80% (B).

McCaffrey et al., 2002

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RNAi in vivo

• Endogenous inhibition

– Sequence specific efficacy of anti-Fas siRNA protects mice

against induced liver failure. Three treatments of 2mg/kg siRNA

administered over a 24 h lead to a reduction of hepatic Fas

mRNA persisting up to 10 days. This was complemented by

lowered hepatic apoptosis and increased survival [Song et al.,

2003. RNA interference targeting Fas protects mice from

fulminant hepatitis. Nat. Med. 9:347-351]

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In vitro transcription

• In vitro transcription of two siRNA strands from DNA oligonucletide templates.

• These templates consist of a sequence complementary to the T7 RNA polymerase promoter followed by a sequence complementary to either the sense or antisense siRNA strand.

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In vitro transcription

• A second oligonucleotide consisting of the T7 promoter coding sequence was annealed;

• The T7 region becomes double stranded (fill-in by Klenow +dNTP)

• The sense or antisense siRNA strand by T7 RNA are transcribed by T7 polymerase.

• The two strands are hybridized to form the siRNA.

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shRNA

• Synthetic siRNAs have short half lives once transfected.

• Eukaryotic polymerase III (Pol III) promoters can be used to express siRNA.

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Expression by Pol III

• A sequence coding for the sense strand of the siRNA of interest, followed by a spacer and then the equivalent to the anti-sense strand, which ends in a series of 5 U residues.

• The spacer mediates the formation of a hairpin structure, allowing the sense and antisense to form base pairs.

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Expression by Pol III

• Eurkaryotic H1 and U6 Pol III promoters are used to drive the expression of the shRNA because:– They initiate from position +1 of the transcripts, so no inhibitory

5’ nucleotide sequences are formed.– Transcripts do not terminate with a poly-A tail but with a series of

four to five thymidine residues, resulting in a series of 3’U residues.

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Alternatively

• The two strands can be transcribed separately from two U6-based transcription cassettes included in the same or different vectors; then they are annealed. This is less potent than the hairpin construct.

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Optimal cassette design

• A nine base-long loop sequence mediates an optimal transcript expression; and the loop sequences are processed and not present in the final siRNA.

– The cytomegalovirus (CMV) enhancer sequence either upstream or downstream of the U6 promoter providing increased efficiency of silencing than the unmodified U6.

• Inducible system: The Tet operator (tet-O) sequence is used to replace a non-essential part of the H1 promoter, stably transfecting the cells; transcription depends on the presence of tetracycline.

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Viral delivery

• Expression vectors– Plasmid and viral vectors in the form of

short hairpin RNAs (shRNA) that are subsequently processed by Dicer. This enables both transient and stable transfection.

– Inducible (tetracycline) plasmid vectors and ecdysone inducible retroviral vectors.

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An example

• Neuronal expression or long term expression.– Adenoviral vectors expressing 64 nt p53

siRNAs from Pol III promoters used to reduce the level of p53 in MCF-7 breast cancer and A549 lung cancer cell lines (Shen et al., 2003).

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Adenoviruses

• Adenoviruses consist of a linear dsDNA genome (~35 kb) surrounded by a nonenveloped capsid.

• internalized via clathrin-mediated endocytosis following association with a cellular receptor, e.g., coxsackievirus and adenovirus receptor (CAR) and the integrin alpha-gamma- ß1 receptor.

• The efficiency of infection depends on the number of receptors on host cells.

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Life cycle of adenovirus

• E1 (immediate early) genes have regulatory functions and stimulate the transcription of the E2, E3, and E4 (early) genes.

• E2 proteins are involved in viral replication.• E3 proteins are responsible for evading the host

immune response and for mediating cell lysis after adenoviral particle assembly.

• Later, major and minor transcripts are produced to complete viral particle production.

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Adenoviral constructs

• E1/E3 deletions prevent the virus from replicating in normal cells.

• A special cell line that expresses E1 proteins, HEK 293 cells, permits virus propagation.

• A shuttle vector is used to clone the desired gene.• The shuttle vector with the desired clone (siRNA) is transfected

with a backbone vector into HEK 293 cells.– Backbone vector has an overlapping sequence with the shuttle vector

and includes the majority of the adenoviral genome (minus the E1 and E3 regions),

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siRNA expressing adenovirus shuttle vector pShuttle-H1

• Stem-loop producing oligonucleotides cloned into the Bgl II and Hind III sites.

• The insert is confirmed with EcoRI digestion ( compare 360 bp band of a positive clone; 300 bp band of an empty vector).

• Polymerase III-dependent H1-RNA promoter drives the expression of 19 bp stem-9 nt loop RNA which is processed into functional siRNA by cellular enzymes.

Shen et al., 2003; FEBS Letters 539:111-114

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A recombinant adenovirus expressing p53 shRNA

• pShuttle-H1-p53 linearized and cotransformed into E. coli BJ5183 cells with pAdEasy-1 adenoviral backbone plasmid.

• Recombinants (AdH1-p53) were selected for kanamycin resistance and confirmed by PacI digestion.

• AdH1-p53 was linearized with PacI and transfected into AD-293 packaging cell line to produce recombinant adenovirus.

Shen et al., 2003; FEBS Letters 539:111-114

•The shuttle vector with the desired clone (siRNA) is transfected with a backbone vector into HEK 293 cells.

–Backbone vector has an overlapping sequence with the shuttle vector and includes the majority of the adenoviral genome (minus the E1 and E3 regions),

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Validation by Western Blot• Proteins extracted from cells and p53 levels checked by Western blot. The lower actin bands served as internal

control for equal total protein loading and specificity control.• In AdH1-p53 but not AdH1-empty infected MCF-7 cells, p53 gene was efficiently silenced both 48 and 72 h after

infection

Shen et al., 2003; FEBS Letters 539:111-114

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Advantages/Disadvantages• efficient transduction of many different cell types, e.g.,

terminally differentiated cells.• results in higher levels of RNA expression.• Infection is independent of cell cycle, so express RNA in both

dividing and non-dividing cells. • Integration into host genome is rare, so little chance of

insertional mutagenesis. • recombinant adenoviruses elicit an immune response in

animal systems, so appropriate only for transient RNA expression no gene-therapy.

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THE END