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�eneral Introdu�tion

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General Introduction

miRNAsThe miRNA lin-4 was the first small RNA to be characterized 1. It was discovered as a post-transcriptional regulator of lin-14 expression in the nematode Caenorhabditis elegans. lin-4 binds to the 3’ UTR of the lin-14 mRNA and is essential for the temporal control of postembryonic development. A similar case was described later with the let-7 miRNA, which regulates lin-41 expression and is also important for developmental timing in C. elegans 2. Initially seen as an oddity of nematodes, post-transcriptional regulation of gene expression by miRNAs turned out to be conserved in all animals. There are also miRNAs found in plants and even in viruses, but it is still debated whether plant and animal miRNAs evolved independently. miRNAs are now seen as an abundant class of 19 to 24 nucleotide small regulatory RNAs with important roles in development and disease. The importance of miRNA regulation is underlined by the finding that many miRNAs are conserved to the nucleotide between evolutionary distant species. While the knockout of individual miRNAs has often little phenotypic effect, the knockout of all miRNAs is lethal 3.

miRNA biogenesismiRNAs are expressed endogenously. miRNA genes can reside in intergenic regions, in exons annotated to noncoding RNAs or in introns. Primary transcripts (pri-miRNAs) are generated mostly by RNA polymerase II and are capped and poly-adenylated 4, 5 (Figure 1). pri-miRNAs fold to complex hairpin structures that are recognized by a nuclear protein complex containing Drosha and Pasha (also called DGCR8) 6-9. Drosha contains two RNase III domains, and Pasha is an RNA-binding protein. The latter recognizes ss-/dsRNA junctions in the pri-miRNA and positions Drosha on the substrate 6. Drosha cleaves the pri-miRNA about 11 nt away from the ss-/dsRNA junction and releases a pre-miRNA stem-loop structure with a 2 nt 3’ overhang. This pre-miRNA is exported to the cytoplasm by the Exportin-5 complex 10, 11. In the cytoplasm, the pre-miRNA is processed by a complex containing Dicer 12-14. Dicer is a multidomain protein containing an N-terminal DExH-box helicase domain, a PAZ domain, two RNase III domains and a C-terminal dsRNA-binding domain. The two RNase III domains are used to cleave the pre-miRNAs into miRNA duplexes with two nucleotide 3’ overhangs in an Mg2+ dependent process 15, 16. Consistent with processing by an RNase III enzyme, miRNAs have 5’ mono-phosphate and 3’ hydroxyl ends. In most organisms, Dicer is accompanied by a RNA-binding protein. TRBP and PACT associate with mammalian Dicer, and R2D2 and Loquacious are Dicer partners required for siRNA and miRNA processing, respectively, in Drosophila 17-21. In C. elegans, RDE-4, the only R2D2 homologue characterized so far, is required for the RNAi pathway (see below) but not for miRNA processing 22. Subsequent to dicing, miRNA duplexes are handed over to the RNA-induced silencing complex (RISC) 23, 24. Core component of RISC is an Argonaute

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protein, which binds the miRNA and is crucial for downstream steps of the silencing process. After RISC loading, the mature miRNA is retained, whereas the other strand of the duplex, the miRNA star (miRNA*) is discarded. The Argonautes required for the miRNA pathway in C. elegans are ALG-1 and ALG-2 12.

Argonaute associationAs for most silencing processes involving small RNAs, an Argonaute protein is essential for miRNA-mediated silencing 12, 25, 26. Argonaute proteins bind small RNAs and in many cases perform enzymatic reactions to down-regulate target RNAs 27-31. They form a diverse protein family with a varying number of members in different organisms 31. Mammals carry seven, Arabidopsis ten, S. pombe one and C. elegans as many as 27 Argonaute genes. Three clades are distinguished in the family tree of Argonautes. Members of the AGO clade (which includes ALG-1 and ALG-2) are in most organisms required for miRNA-mediated silencing and RNAi (see

Figure 1 Model of the miRNA pathway in C. elegans. miRNA genes (e.g. miR-35) are transcribed by RNA Pol II. The transcripts fold into complex secondary structures (pri-miRNA). These structures are cropped by a complex containing Drosha (DRSH-1) and Pasha (PASH-1) into hairpin precursors (pre-miRNA), which are exported from the nucleus. In the cytoplasm, Dicer (DCR-1) cleaves the pre-miRNAs and releases the mature miRNA, which is bound by the Argonaute proteins ALG-1 or ALG-2 and guides them to the target mRNA. Upon targeting, translation is inhibited, or the mRNA is de-adenylated and degraded.

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below). Members of the Piwi clade bind the recently discovered class of piRNAs and functions in transposon silencing in the germline 32-34. There is an additional worm-specific clade with mostly unknown function. Some members of this clade have recently been shown to bind secondary siRNAs 35. Most Argonaute proteins carry out specialized functions, although some family members also show redundancy. Argonaute proteins are defined by two conserved domains: The PAZ domain (named after three Argonaute protein family members Piwi, Argonaute and Zwille), and the PIWI domain. Solving the crystal structures of Argonaute proteins from different (bacterial) species revealed the function of these two domains. The PAZ domain provides a binding pocket for the 3’ end of the small RNA 36-41, while the PIWI domain binds the 5’ end of the small RNA, leaving the nucleotides available for base pairing to the target RNA 42. When a small RNA binds to a target RNA, the target RNA is positioned into a crescent over the PIWI domain 43, 44. The PIWI domain also contains homology to RNase H. In many Argonaute proteins, this RNase H motif is active and can cleave dsRNA opposite to nucleotides 10 and 11 of the small RNA, mediated by three conserved catalytic residues (in most cases DDH) 25, 44-48. For miRNA-mediated silencing, however, such a cleavage of the target RNA is the exception 24, 26, 49. In most cases, animal miRNAs base pair to the target mRNA with imperfect complementarity, avoiding an Argonaute-mediated cleavage. Base pairing between the seed region of the miRNA (nt 2-8) has been found to be crucial for function, although extensive complementarity of the 3’ part of the miRNA can also have an influence on silencing efficiency 50-52.

miRNA silencing mechanismmiRNA targets are mostly localized to the 3’ UTR of messenger RNAs, where the sequence context may also influence the efficiency of target site recognition 53-55. Most miRNA have multiple target sites, and several different miRNAs often target the same 3’UTR, acting cooperatively in silencing 56. The exact mechanism of silencing is still debated. Most studies report an effect of translation inhibition, either by preventing translation initiation or translation elongation 50, 57-61. Other studies have found evidence for de-adenylation to promote degradation 62, 63. In most cases, repressed mRNAs are localized to P-bodies, cytoplasmic foci where mRNA is de-capped and degraded 64-68.

miRNA discoveryThe first miRNAs (lin-4, let-7, lsy-6) were discovered in genetic screens 1, 2, 69. However, this turned out not to be the ideal way to identify more miRNAs because of their small size and the fact that the knockouts of most miRNAs have only very subtle phenotypes 70. Once it was realized that miRNAs are abundant, cloning efforts were undertaken to determine the complete set of miRNAs in various organisms and tissues 71-84 (chapter two). These cloning efforts are still ongoing, as deep sequencing facilitates the process and allows the discovery of very low abundant miRNAs 85. In parallel,

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computational approaches were taken to find novel miRNAs. Algorithms using combination of secondary structure prediction and conservation are useful to predict at least conserved miRNAs, although experimental validation is still crucial 77, 86-89. The exact number of miRNAs is still debated, especially because it is not clear how a miRNA is defined. The consensus at the moment is that a miRNA is a small RNA of 18 to 24 nucleotides that needs to be expressed from a hairpin precursor and processed by Dicer, and ideally both strands of the miRNA-duplex should be detectable 90. However, this definition does not include functionality, and many low abundant miRNAs might not be biologically relevant. An updated list of all miRNAs is available at miRBase (http://microrna.sanger.ac.uk). Small RNAs that are associated with ALG-1 and ALG-2 may function as miRNAs in vivo, a feature that we exploit for miRNA discovery in chapter three.

RNAiThe second class of small RNAs discovered was small interfering RNAs (siRNAs). Andrew Fire, Craig Mello and colleagues discovered that dsRNA induces potent down regulation of mRNAs of homologous sequence 91, and it was soon realized that this mechanism, termed RNA interference (RNAi) involved small RNAs 92, 93. The process is conserved in animals and plants, with differences especially in the downstream steps of the pathway. Because the thesis focuses on RNA silencing in C. elegans, the following paragraph mostly describes RNAi in C. elegans.

siRNA biogenesissiRNA biogenesis, like miRNA biogenesis, involves a Dicer protein family member 94 (Figure 2). While flies and plants have separate Dicers for the miRNA- and the siRNA-pathway, mammals and nematodes use the same protein 13, 14, 29, 95-97. After dicing, siRNA duplexes are loaded into the RNA-induced silencing complex (RISC) 17, 23. Dicing and RISC loading are linked, and in some organisms, an intermediate RISC-loading complex (RLC) that is distinct form the Dicer complex and RISC can be purified 98, 99. Only one strand of the siRNA duplex (the guide strand) is retained in RISC, the other strand (the passenger strand) is eliminated. Thermodynamic stabilities within the duplex determine strand selection. The two-nucleotide 3’-overhang of the end of the duplex with weaker base pairing properties is bound by the PAZ domain of the Argonaute protein in RISC, and the bound strand becomes the guide strand. This process is best understood in Drosophila, where the R2D2, a dsRNA-binding protein that interacts with Dcr-2, acts as a sensor for siRNA duplex asymmetry 100. RDE-4, the C. elegans homolog of R2D2, also stably interacts with Dicer, and may have similar function 22.

Argonaute associationMost organisms have separate Argonaute proteins for the different silencing pathways 31. The Argonaute required for RNAi in C. elegans is RDE-1 101 (Figure 2).

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Following Argonaute loading of the siRNA duplex, the passenger strand is cleaved by the RNase H activity in the PIWI domain 45, 102, 103. If cleavage is prevented by mismatches within the siRNA duplex, a second, slower process can separate the two strands by a mechanism that most likely involves a helicase 103. The retained guide strand acts as a guide molecule to tethers RISC to target RNAs by homologous base pairing. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Good accessibility of the target site is essential for this process, as RISC is unable to unfold structured RNA 104. In most organisms, the target RNA is cleaved opposite to the bond between nucleotide 10 and 11 of the siRNA by the RNase H activity in the PIWI domain, and subsequently degraded by exonucleases 44, 46, 48, 105,

106. For vertebrates and insects, the RISC-mediated target cleavage is sufficient to down-regulate target RNA levels. It is, however, doubtful whether C. elegans RNAi involves a RISC-mediated cleavage if the target RNA at this step (chapter six).

Figure 2 Model of RNAi in C. elegans. Long double-stranded RNA (dsRNA) is cleaved by Dicer (DCR-1) into small interfering RNA duplexes (siRNAs). Dicing of siRNAs is coupled to loading them into the Argonaute protein RDE-1. Upon loading, the passenger strand of the siRNA duplex is discarded, and the guide strand remains bound in RDE-1 to form the RNA-induced silencing complex (RISC). RISC is guided to the target RNA by the siRNA and recruits an amplification machinery including the RNA-dependent RNA polymerase RRF-1. RRF-1 generates secondary siRNAs, possibly with the aid of DCR-1. Secondary siRNAs are bound by secondary Argonautes, which are required for target degradation and silencing.

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AmplificationIn nematodes, plants and fungi, an amplification step is required to achieve efficient silencing via RNAi 107-112. This amplification step involves an RNA-directed RNA polymerase (RdRP) that generates secondary siRNAs using the target RNA as template 109 (Figure 2). There are four RdRP genes in C. elegans, six in Arabidopsis and one in S. pombe. C. elegans RRF-1 is required for somatic RNAi 109. EGO-1 is very similar to RRF-1 (the two proteins are functionally interchangeable) and required for germline RNAi 110. In addition to that, it has functions in germline development and maintenance 113. The function of RRF-2 is not well defined, but it seems to be involved in germline RNAi in some cases (R. Plasterk and B. Tops, in preparation). RRF-3 has been identified as an enhancer of (exogenous) RNAi and functions in the endogenous RNAi pathway 114. Recent studies have revealed details about the initiation of secondary siRNA production (chapter four) 115. However, the biochemistry of RdRP recruitment and fuction is still not fully understood. The model predicts that upon target recognition by RISC, the RRF-1 complex is recruited to the target site to generate secondary siRNAs. In C. elegans, these secondary siRNAs are found only upstream of the primary trigger and are only of antisense polarity relative to the target 109. Secondary siRNAs are bound by different Argonaute proteins than primary siRNAs. In C. elegans, they are bound by at least four nematode-specific family members termed secondary Argonautes that have redundant function in RNAi 35 (appendix to the introduction). These Argonaute proteins are involved in target down-regulation, as secondary siRNAs have been shown to act in trans 109, and the secondary Argonaute knockout line is RNAi-deficient 35. Whether this degradation also involves an RdRP or is achieved through a different mechanism remains to be determined.

RNAi as a tool in researchThe discovery of RNAi has not only revealed an additional layer of post-transcriptional regulation of gene expression, it has also provided tools that revolutionized research. RNAi is now widely used to knock down genes and to study loss of function phenotypes. Different genome-wide RNAi libraries are available for C. elegans 116-

118. Long dsRNA triggers corresponding to every gene in the genome can be delivered by expressing them in bacteria on which the nematodes feed. The dsRNA is taken up through the gut 119, 120, and the silencing signal can spread through the whole organism (systemic RNAi) 121. The endogenous amplification machinery can use a few trigger molecules to establish a robust silencing response. Genome-wide dsRNA libraries for Drosophila cell cultures and tobacco and Arabidopsis libraries in viruses were developed as well 122-125. Mammalian cells do not tolerate long dsRNA due to the interferon response. This problem was tackled by developing short hairpin RNA libraries, which made genome wide RNAi screens possible also for mammalian cell cultures 126-128.

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Other RNA silencing pathways and classes of small RNAsRNAi and the miRNA pathway are seen as the main examples of pathways involving small RNAs, but there are a number of other silencing pathways that function through small RNAs as well. A phenomenon called co-suppression was observed in plants even before RNAi was discovered 129, 130. Additional copies of a gene can induce silencing of the gene by a mechanism that is related to, but not identical with RNAi and also seems to have a transcriptional component. A similar phenomenon is also observed in the germline of C. elegans 131, 132. Genetic screens identified a number of genes required for co-suppression, some of which are also required for RNAi 133. In yeast, the RNA silencing machinery is mainly used for silencing through heterochromatin formation 111. Small RNAs are generated by Dicer, and the RNA-induced transcriptional silencing (RITS) complex interacts with the RdRC complex to recruit a machinery that induces heterochromatin formation 134-136. In fact, this silencing does not appear to be entirely transcriptional, as low amounts of transcripts are still generated, and the process is now called co-transcriptional silencing 137. Heterochromatin formation also occurs in plants 138-141. There have been observations of RNA induced transcriptional silencing in Drosophila, C. elegans and mammals, but these cases are less well characterized 142-147. RNAi is seen as a defense mechanism against viruses 148-152, and it is therefore not surprising that suppression of transposable elements is also mediated by small RNAs 101, 153-156. Initial findings came from genetic screens performed in C. elegans and Drosophila, although they offered limited mechanistic insight. In C. elegans, there is evidence for a model that predicts snap-back loops of the transposon transcript and the local formation of dsRNA, which is recognized by part of the RNAi machinery 157. As in the case of co-suppression, many genes involved in transposon silencing are also required for RNAi 101, 154, 158. In flies and vertebrates, there is a separate class of small RNAs, the piRNAs (Piwi-associated RNAs), that protects the germline against transposition 32, 33, 153, 159-161. These small RNAs are longer than miRNAs and siRNAs (24-26 nt in Drosophila, around 30 nt in vertebrates). Their biogenesis does not involve Dicer cleavage, and they function through association with the Piwi clade members of Argonautes 33, 34, 162, 163. Small RNAs are also involved in mechanism in Tetrahymena in which the genomic content of nuclei are compared within a shared cytoplasm prior to chromatin modification and targeted DNA elimination 164-167.The variety of mechanisms involving small RNAs has not yet been fully uncovered. In C. elegans alone there are a number of classes of small RNAs with completely unknown function. These include small endogenous RNAs (endo-siRNAs), tiny non-coding RNAs (tncRNAs) and 21U RNAs 74, 85. Based on their end modifications, most endo-siRNAs analyzed so far are probably products of an RdRP activity. Their biogenesis requires RDE-4, RDE-2, MUT-7, ERI-1/3/5, RFF-3, DRH-3 and the Argonaute protein ERGO-1, and secondary endo-siRNAs bind to the same set of secondary Argonautes as secondary siRNAs produced during exo-RNAi 35, 168,

169. 21U RNAs are expressed from a cluster on chromosome 2 and appear to be

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individually driven by a promoter sequence 85. Most of the different pathways involving small RNAs have overlapping genetic requirements, but are functionally distinct. More research will be required to fully understand the whole small RNA silencing network in C. elegans and other organisms.

Open questionsThe Dicer, Argonaute and RdRP proteins and their functions in the RNAi pathway are relatively well understood. However, there are a number of aspects that remain unclear. In most organisms, RNAi relies on the RNase H activity in the Argonaute protein. Based on sequence alignments, RDE-1 carries the residues required to form the RNase H motif, but cleavage activity has not been found for RDE-1, notwithstanding numerous attempts by several labs. It is also not clear how RRF-1 is recruited to the target RNA, and how secondary siRNAs are generated. The current model predicts that RRF-1 generates an antisense transcript along the target mRNA, and that the resulting dsRNA is cleaved by Dicer. However, C. elegans secondary siRNAs are exclusively of antisense polarity and carry 5’ triphosphates, arguing against a role of Dicer in their processing (chapter four) 109, 115. There are a number of additional factors required for RNAi, which have been identified in genetic screens for RNAi-deficient (rde) mutants 154, 158, 170, 171. Examples are rde-2 (novel protein), rde-3 (polyA polymerase), mut-7 (RNaseD homolog), mut-14 (ATP-dependent RNA helicase), mut-15 (unknown), mut-16 (glutamine/asparagine (Q/N)-rich domain-containing protein). Although there are a limited number of follow-up studies 172-174, these factors are difficult to place within the pathway, and their exact role is unknown. The precise endogenous function of RNAi is still not fully understood. Many of the rde mutants (rde-1, rrf-1, rde-4) have no phenotype except RNAi deficiency. It is likely that RNAi is used to defend the genome against viruses. The role of RNAi in virus defense is well established in plants and has been demonstrated for C. elegans under artificial conditions 148, 149, 151. However, no natural nematode viruses have been found to date. There are multiple pathways involving different classes of small RNAs in C. elegans, and the number of Argonaute proteins implies that there are more to be found. Since small RNA-pathways intersect (e.g. on Dicer or on secondary Argonautes 35, 169), it is puzzling and intriguing how the pathways are kept separate, and how specificity of the different silencing processes is achieved.

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Outline of this thesis

miRNAs were the first small RNAs to be discovered. They are important regulators of gene expression in development and are often expressed in a tissue specific way. Cataloguing the miRNA repertoire and analyzing the expression profiles may therefore give insight in miRNA function. Chapter two describes a cloning and expression analysis of novel miRNAs from zebrafish.

Most small RNAs function through Argonaute association. ALG-1 is an Argonaute protein that is required for the miRNA pathway in C. elegans. In chapter three, we show that ALG-1 almost exclusively binds miRNAs. We present ten novel miRNAs and propose Argonaute association as an additional criterion for miRNA definition.

In chapter four, we show that in C. elegans, secondary siRNA biogenesis does not involve priming of the primary siRNA. We also show that secondary siRNAs carry 5’ triphosphate modifications, are not bound by RDE-1 and thus represent a separate class of small RNAs.

In C. elegans, siRNAs and miRNAs are both produced by the same Dicer protein, but function through different pathways. In chapter five, we show that there are features in the structure of the double stranded precursor that drives a small RNA into the RNAi- or the miRNA pathway.

In chapter six, we review and discuss the key findings of this thesis and show preliminary results regarding the role of RDE-1 in RNAi and related pathways.

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25

Appendix to the �eneral Introdu�tion

Kno�king out the Argonautes

reprinted from Cell, 127:667-8 (2006)

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27

�eneral Introdu�tionLeading Edge

Previews

Cell 127, November 17, 2006 ©2006 Elsevier Inc. 667

The finding that double-stranded(ds)RNAs can induce gene silencingthrough RNA interference (RNAi) ata posttranscriptional level is a mile-stone discovery (Fire et al., 1998) thatgarnered this year’s Nobel Prize inPhysiology or Medicine. The under-lying mechanism of RNAi is remark-ably conserved and relatively simple.dsRNA of an endogenous or exog-enous source is cut into small inter-fering RNA (siRNA)-duplexes by theRNase III enzyme Dicer (Bernstein etal., 2001). Dicing is coupled to loadingthese siRNAs into the RNA-inducedsilencing complex (RISC), which hasan Argonaute protein as the coreenzyme. The siRNA guides the RISCcomplex to the target messenger RNA(mRNA), which is degraded throughendonucleolytic cleavage by the Arg-onaute protein (Liu et al., 2004).

Argonaute proteins are character-ized by two domains. The PAZ domainprovides a binding pocket for the 3!end of the siRNA, the PIWI domainbinds to the 5! end of the siRNA as wellas to the target RNA and carries activeresidues for endonucleolytic cleavagein many, but not all, family members.The number of Argonaute protein fam-ily members varies between organ-isms: humans have eight, Drosophilafive, Arabidopsis ten, and the wormCaenorhabditis elegans as many as27. Although it is likely that all Argo-nautes bind to small RNAs, only afew have been placed into one of themany RNA silencing pathways, andfew of the classes of small RNAs towhich they bind have been character-ized. In this issue, Yigit et al. (2006)provide a comprehensive analysis ofthe C. elegans Argonautes, knock-

ing out all 27 worm Argonaute genes.They describe the function as well asthe small RNA partners for a numberof previously uncharacterized familymembers. Perhaps their most intrigu-ing findings are that several differentArgonautes act at different steps in thesame RNAi pathway and that differentRNA silencing pathways intersect on aspecific class of Argonautes.

WormRNAidiffers fromRNAi inmostother animals because the silencingsignal is amplified (Sijen et al., 2001).The siRNAs produced from the initial(exogenous) dsRNA trigger are calledprimary siRNAs, and the Argonautethat binds to these (RDE-1) is calleda primary Argonaute. Once RDE-1 isguided to the target RNA by the pri-mary siRNA, an RNA-directed RNApolymerase (RdRP) generates newdsRNAs upstream of the initial trigger.The new dsRNAs are processed intoa new class of siRNAs, called second-ary siRNAs, which can initiate anotherround of silencing. One biologicalrole of the worm RNAi machinery isto defend the cell against molecularparasites—such as viruses and trans-posons (Plasterk, 2002)—which needto be constantly silenced. In a perma-nent effort to discriminate self fromnonself, it is crucial to ensure the spe-cificity of the amplified signal. Ampli-fication ensures the efficient removalof target RNA, but it also leads to anincreased risk of nonspecific or “off-target” effects by the perpetual gener-ation of secondary siRNAs. Several ofthe Argonautes that Yigit et al. (2006)describe (SAGO-1, SAGO-2, and PPW-1) bind to and stabilize secondary siR-NAs and are therefore called second-ary Argonautes. They are required for

RNAi, they are redundant (only wormslacking the group of secondary Argo-naute-encoding genes are completelyRNAi deficient), and they seem to formthe rate-limiting step of the process,as their overexpression leads to accu-mulation of more secondary siRNAs.Interestingly, they lack the conservedcatalytic residues required for tar-get cleavage. Their inability to cleavetarget RNAs may prevent them frominducing further amplification and theexponential generation of second-ary siRNAs. However, their require-ment for silencing shows that they areinvolved in the RNA target degrada-tion that accompanies RNAi througha mechanism that has yet to be deter-mined. Another open question is howthe pathway distinguishes primary andsecondary siRNAs in order to be ableto load them into distinct Argonautes,given that both classes of small RNAsare processed from dsRNA precur-sors. The RdRP complex might recruitboth Dicer and specifically secondaryArgonautes; dsRNA generation, dic-ing, and Argonaute loading might thusbe coupled.

RNAi is not the only posttranscrip-tional silencing pathway that involvessmall RNAs. The Argonautes that arerequired for silencing by microRNAsin worms have been characterized(Grishok et al., 2001). Another Argo-naute described by Yigit et al. (2006),ERGO-1, is required for production ofendo-siRNAs, a class of small RNAsof unknown function first describedby Ambros et al. (2003). Based onhomology, ERGO-1 is likely to actas a primary Argonaute in the endo-RNAi pathway, analogous to RDE-1in the exo-RNAi pathway. It remains

Knocking out the Argonautes

Florian A. Steiner1 and Ronald H.A. Plasterk1,2,*1Hubrecht Laboratory—KNAW, Utrecht, 3584 CT, The Netherlands2Utrecht University, Utrecht, 3508 TC, The Netherlands*Contact: plasterk@niob.knaw.nlDOI 10.1016/j.cell.2006.11.004

Argonaute proteins are key players in gene silencing involving small RNAs. In this issue,Yigit et al. (2006) report a comprehensive study of Argonautes in the worm that placesmany of the 27 family members into a complex gene-silencing network.

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to be elucidated how primary siRNAsfrom different sources end up in dif-ferent Argonautes after dicing. Inter-estingly, the secondary Argonautesthat bind to secondary exo- siRNAsalso bind to endo-siRNAs, implyingthat endo-RNAi is also an RdRP-amplified process. In any case, theexo- and endo-RNAi pathways seemto converge on the secondary Argo-nautes. They might thus represent themain degrading mechanism of targetRNAs, which is shared between thetwo RNAi pathways, with the primaryArgonautes providing primary target

recognition and ensuring specificityof the amplification (Figure 1).

The range of Argonaute proteinfunction seems to go beyond post-transcriptional gene-silencing path-ways. In Drosophila and mammals,the PIWI Argonautes act in the germ-line and bind to distinct classes ofsmall RNAs (reviewed in Kim, 2006).Argonautes are also involved in chro-matin remodeling in many organisms,which has been most studied in yeast(Verdel et al., 2004). In C. elegans, asin fungi, RNAi can induce heritablegene silencing, probably at the level of

chromatin (Vastenhouw et al., 2006),and it is possible that Argonautes arealso involved in this process. Yigit etal. (2006) describe a worm Argonauteknockout (csr-1) with chromosomesegregation defects at metaphaseof the cell cycle in the early embryo.Another Argonaute knockout (prg-1)exhibited reduced fertility and a tem-perature-sensitive sterile phenotype.

Although we do not yet understandthe molecular mechanisms of theseprocesses, all Argonautes analyzedso far share their ability to bind tosmall RNAs. The future challengesare obvious. How many classes ofsmall RNAs exist? Which Argonautesbind to which classes of small RNAs?How are these small RNAs gener-ated? And what are their biologicalfunctions? Yigit et al. (2006) showthat certain Argonautes are involvedin development, fertility, and RNAi,but for many Argonautes, their func-tions remain to be determined.

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Figure 1. The Endo- and Exo-RNAi Pathways Converge on Secondary ArgonautesThe model postulates that primary small interfering (si)RNAs are produced from double-stranded(ds)RNA by Dicer in both the exo- and endo-RNA interference (RNAi) pathway. Primary Argonautesare distinct for the two pathways, with RDE-1 binding to primary exo-siRNAs and ERGO-1 bindingto primary endo-siRNAs. After mRNA target recognition and presumably cleavage by the primaryArgonaute, an RNA-dependent RNA polymerase (RdRP) generates new dsRNA, which acts againas a substrate for Dicer. The resulting secondary siRNAs of both pathways are bound by the sameset of secondary Argonautes, which includes SAGO-1, SAGO-2, and PPW-1. The secondary Argo-nautes are involved in target degradation by an unknown mechanism. (Worm Argonautes charac-terized by Yigit et al. (2006) are shown in color; characterized RNAi factors are gray).