Cell Science at a Glance 1819

Argonaute at a ) – ncRNAs that are characteristically Biogenesis of miRNAs and siRNAs ~20-35 long – are required for miRNAs glance the regulation of expression in many Generally, miRNAs are transcribed by RNA different organisms. Most small RNA species polymerase II or III to form stem-loop-structured Christine Ender1 and Gunter fall into one of the following three classes: primary miRNA transcripts (pri-miRNAs). pri- Meister1,2,* (miRNAs), short-interfering RNAs miRNAs are processed in the nucleus by the 1 Center for Integrated Science Munich (siRNAs) and -interacting RNAs (piRNAs) microprocessor complex, which contains (CIPSM), Laboratory of RNA Biology, Max-Planck- Institute of Biochemistry, Am Klopferspitz 18, 82152 (Carthew and Sontheimer, 2009). Although the RNAse III and its DiGeorge Martinsried, Germany different small RNA classes have different syndrome critical region gene 8 (DGCR8) 2University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany bio genesis pathways and exert different cofactor. The transcripts are cleaved at the stem *Author for correspondence functions, all of them must associate with a of the hairpin to produce a stem-loop- ([email protected]) member of the protein family for structured miRNA precursor (pre-miRNA) of

Journal of Cell Science 123, 1819-1823 activity. This article and its accompanying ~70 nucleotides. After this first processing step, © 2010. Published by The Company of Biologists Ltd poster provide an overview of the different pre-miRNAs are exported into the cytoplasm by doi:10.1242/jcs.055210 classes of small RNAs and the manner in which exportin-5, where they are further processed they interact with Argonaute protein family by the RNAse III enzyme and its TRBP Although a large portion of the members during small-RNA-guided gene (HIV transactivating response RNA-binding is actively transcribed into RNA, less than 2% silencing. In addition, we highlight recently protein) partner. Dicer produces a small double- encodes proteins. Most transcripts are non- uncovered structural features of Argonaute stranded RNA (dsRNA) intermediate of coding RNAs (ncRNAs), which have various proteins that shed light on their mechanism of ~22 nucleotides with 5 phosphates and cellular functions. Small ncRNAs (or small action. 2- 3 overhangs. In subsequent

Argonaute Proteins at a Glance Christine Ender and Gunter Meister

Structure of Argonaute proteins Small-RNA biogenesis and mechanisms of action

Highest Cytoplasm heterogeneity RNA binding Active site Nucleus dsRNA from exogenous sources siRNA from exogenous sources mRNA cleavage N terminus PAZ MID PIWI AGO L1 L2 siRNAs Cofactor Cofactor 3Ј OH-binding site 5Ј P-binding site Repetitive elements, Dicer 7 pseudogenes, other AGO m G AAA Crystal structure of ThermusThermus thermophilusthermophilus ArgonauteArgona endo-siRNA loci AGO RISC

Journal of Cell Science Endo-siRNAs PAZ MID BackboneB phosphorus atomsa are in yellow miRNAs (Reproduced( with Primary permissionp from Drosha miRNA DGCR8 WangW et al., 2008a) transcripts Processing Translational AGO repression and/or N Microprocessor deadenylation TRBP Dicer Pol miRNA L1 II/III precursor AGO m7G AAA AGO PIWI Exportin-5 miRNAs miRNP L2 Splicing Guide-DNA–target-RNA duplex used in crystal structure 110 21 Guide DNA 5Ј p-TGAGGTAGTAGGTTGTATAGT 3Ј Mirtron Spliceosome Primary piRNA generation oo piRNAs Target RNA3Ј UGCUCCAUCACUCAACAUAU 5Ј 1Ј 10Ј 19Ј Repetitive elements, piRNA precursors transposons, piRNA clusters Arginine methylation of Piwi proteins

Sequence motifs MIWI2 MILI MIWI Generation of piRNA for symmetrical GRG 5Ј ends by MILI dimethylarginine GRA MILI PRMT5 MIWI (sDMA) modification, ARG MILI MILI MILI often in repeats MIWI ? TDRD MIWI2 sDMA CH CH 3 3 MIWI2 MIWI modification MILI Transposon silencing HN + NH Secondary processing: C ping-pong model TDRD NH MILI Trimming/cleavage MIWI2 CH of piRNA 3Ј ends 2 TDRD TDRD CH2 MIWI2 CH 2 TDRD MILI MIWI C + – H N H MILI Transposon silencing 3 COO Methylation of Piwi MIWI2 proteins enables their interaction with Generation of piRNA MIWI2 functionally important piRNA precursors 5Ј ends by MIWI2 TDRD = sDMA modification TDRD proteins

Abbreviations: DGCR8, DiGeorge syndrome critical region gene 8; dsRNA, double-stranded RNA; endo-siRNA, as PIWIL1 (Piwi-like homolog 1); MIWI2, also known as PIWIL4 (Piwi-like homolog 4); piRNA, Piwi-interacting RNA; Pol siRNA derived from endogenous source; L1, linker 1; L2, linker 2; miRNA, microRNA; miRNP, miRNA-containing II/III, RNA polymerase II or III; PRMT5, protein arginine methyltransferase 5; RISC, RNA-induced silencing complex; siRNA, ribonucleoprotein particle; Mirtron, miRNA-intron; MILI, also known as PIWIL2 (Piwi-like homolog 2); MIWI, also known small-interfering RNA; TDRD, Tudor-domain-containing protein; TRBP, HIV transactivating response RNA-binding protein. © Journal of Cell Science 2010 (123, pp. 1819-1823)

(See poster insert) 1820 Journal of Cell Science 123 (11)

processing and unwinding steps, one strand of The Argonaute protein family Argonaute proteins typically have a the dsRNA intermediate is incorporated into an To perform their effector functions, small RNAs molecular weight of ~100 kDa and are Argonaute-protein-containing complex that is must be incorporated into Argonaute-protein- characterized by a Piwi-Argonaute-Zwille referred to as an miRNA-containing containing complexes. Argonaute proteins are (PAZ) domain and a PIWI domain. ribonucleoprotein particle (miRNP). The highly specialized small-RNA-binding Crystallographic studies of archaeal and opposite strand, the so-called miRNA* (miRNA modules and are considered to be the key bacterial Argonaute proteins revealed that the ‘star’) sequence, is thought to be degraded. In components of RNA-silencing pathways. PAZ domain, which is also common to Dicer some cases, however, miRNA* sequences can Argonaute proteins were named after an AGO- , forms a specific binding pocket for the also be functional (Packer et al., 2008). Mature knockout phenotype in 3-protruding end of the small RNA with which miRNAs guide Argonaute-containing complexes that resembles the tentacles of the octopus it associates (Jinek and Doudna, 2009). The to target sites in mRNAs that are partially Argonauta argo (Bohmert et al., 1998). On the structure of the PIWI domain resembles that of complementary to the miRNA sequence, and basis of sequence , Argonaute bacterial RNAse H, which has been shown to induce repression of at the level proteins can be divided into two subclasses. cleave the RNA strand of an RNA-DNA hybrid of mRNA stability or (see poster). One resembles Arabidopsis AGO1 and is (Jinek and Doudna, 2009). More recently, it was In addition to this canonical miRNA referred to as the Ago subfamily; the other discovered that the catalytic activity of miRNA biogenesis pathway, some alternative is related to the Drosophila PIWI protein and is effector complexes, also referred to as Slicer miRNA biogenesis pathways have recently been referred to as the Piwi subfamily. activity, resides in the Argonaute protein itself. discovered. So-called mirtrons (miRNA- Members of the human Ago subfamily, which Interestingly, not all Argonaute proteins show introns) are miRNAs found in introns. They are consists of AGO1, AGO2, AGO3 and AGO4, endonucleolytic activity. In , only identical to pre-miRNAs with respect to their are ubiquitously expressed and associate with AGO2 has been shown to cleave the size and features, but are generated miRNAs and siRNAs. Ago proteins are phosphodiester bond of a target RNA, at a site independently of Drosha (Kim et al., 2009; conserved throughout species and many opposite nucleotides 10 and 11 of the siRNA, Siomi and Siomi, 2009). Moreover, some organisms express multiple family members, although the conserved aspartic acid-aspartic classes of small nucleolar RNAs (snoRNAs) can ranging from one in Schizosaccharomyces acid-histidine (DDH) motif (which is important be processed into small RNAs that act like pombe, five in Drosophila, eight in humans, ten for divalent metal ion binding and catalytic miRNAs (Ender et al., 2008; Scott et al., 2009; in Arabidopsis to twenty-seven in C. elegans activity) is also present in human AGO3 (Liu Taft et al., 2009). (Tolia and Joshua-Tor, 2007). Argonaute et al., 2004; Meister et al., 2004). proteins are also present in some species of More recently, structural studies have been siRNAs budding yeast, including Saccharomyces extended to Thermus thermophilus Argonaute in There are many similarities between the castellii. It was recently found that S. castellii complex with a guide strand only or a guide pathways by which miRNAs and siRNAs are expresses siRNAs that are produced by a Dicer DNA strand and a target RNA duplex. This processed. In contrast to miRNAs, however, protein that differs from the canonical analysis revealed that the structure of the siRNAs are processed independently of Drosha Dicer proteins found in animals, plants and other complex is divided into two lobes. One lobe from long dsRNAs that are derived from fungi (Drinnenberg et al., 2009). However, the contains the PAZ domain connected to the

Journal of Cell Science either exogenous sources (to form exo- model organism Saccharomyces cerevisiae N-terminal domain through a linker region, L1. siRNAs) or endogenous sources (to form lacks Argonaute proteins and none of the known The second lobe consists of the middle (MID) endo-siRNAs). Depending on the organism, small RNA pathways are conserved in domain (located between the PAZ and the PIWI exo-siRNAs can be produced through S. cerevisiae. Argonaute proteins are also found domains) and the PIWI domain. The 5 the cleavage of viral RNAs or by in some prokaryotes (Jinek and Doudna, 2009), phosphate of the small RNA to which Argonaute introducing long, perfectly base-paired dsRNA but their function in these organisms remains binds is positioned in a specific binding pocket into the cytoplasm. Both Dicer and TRBP are unclear. in the MID domain (Jinek and Doudna, 2009). required for the processing of siRNAs from The expression of Piwi subfamily members is The contacts between the Argonaute protein and long dsRNAs that are derived from exogenous mainly restricted to the germline, in which they the guide DNA or RNA molecule are dominated sources. Endo-siRNAs have been reportedly associate with piRNAs. The by interactions with the sugar-phosphate expressed in plants and in animals such as encodes four Piwi proteins, named HIWI (also backbone of the small RNA or DNA; thus, the Caenorhabditis elegans, flies and mice. known as PIWIL1), HILI (also known as bases of the RNA or DNA guide strand are free Endo-siRNAs are derived from transposable PIWIL2), HIWI3 (also known as PIWIL3) and for base pairing with the complementary target elements, natural antisense transcripts, long HIWI2 (also known as PIWIL4) (Peters RNA. The structure indicates that the intermolecularly paired hairpins and and Meister, 2007). There are three Piwi target mRNA base pairs with the guide DNA pseudogenes (Czech et al., 2008; Ghildiyal proteins in mice, known as MIWI, MILI and strand, at least in the region of the seed sequence et al., 2008; Kawamura et al., 2008; MIWI2. (which is especially important for target Okamura et al., 2008). recognition), but does not touch the protein After Dicer processing, siRNA duplexes are Structure and function of Argonaute (Wang et al., 2008a; Wang, Y. et al., 2009; Wang separated and one strand is incorporated into the proteins et al., 2008b). RNA-induced silencing complex (RISC). The Small RNAs regulate gene expression by Although significant progress has been single-stranded RNA, often referred to as guiding Argonaute proteins to complementary made regarding the structure of bacterial and the guide strand (the other strand is known sites on target RNA molecules. Recent structural archaeal Argonaute proteins, no structures of as the passenger strand), directs RISC to studies of bacterial and archaeal Argonaute mammalian Argonaute proteins are available. perfectly complementary sites in target mRNA proteins have shed light on the mechanism of As bacterial Argonaute proteins have a high molecules; RISC then cleaves the target silencing mediated by both Ago and Piwi affinity for short DNA guide strands, it will mRNA (Kim et al., 2009). subfamily proteins. be interesting to see how mammalian Journal of Cell Science 123 (11) 1821

Argonaute proteins interact with short RNA (P-bodies), cytoplasmic regions enriched for helped to further elucidate the function of these guide strands. proteins involved in mRNA turnover (Eulalio proteins (Aravin and Hannon, 2008). In miRNAs and siRNAs guide Ago proteins to et al., 2007). Insects have one GW182 protein, , piRNA expression changes during their target mRNA. A key determinant of the C. elegans has two (AIN1 and AIN2) and stages of sperm development. During the pre- regulatory mechanism of RNA silencing is humans have three paralogs (TNRC6A/GW182, pachytene stage of meiosis, piRNAs the degree of complementarity between the TNRC6B and TNRC6C) (Eulalio et al., 2007). predominantly correspond to repetitive and small RNA and the target mRNA. Perfect GW182 proteins associate with Ago proteins transposon-rich sequences, and interact with complementarity promotes AGO2-mediated through direct protein-protein interactions. It Piwi subfamily members MILI and endonucleolytic cleavage, whereas mismatches has been demonstrated that some GW motifs MIWI2. piRNAs found during the pachytene in the central region of the small RNA lead to form Ago-interaction platforms that interact stage of meiosis associate with Piwi subfamily repression of gene expression at the level of with the MID domain of Ago proteins. Such members MILI and MIWI (Aravin and Hannon, translation or mRNA stability. However, the GW repeats are referred to as Ago ‘hooks’ (Till 2008). In mice, it has been demonstrated that mechanisms underlying translational repression et al., 2007). In Drosophila, depletion of piRNAs act together with their respective Piwi are not yet clear. Studies from several GW182 leads to increased levels of mRNAs protein partners to repress the expression of laboratories have provided support for the targeted by miRNAs (Behm-Ansmant et al., mobile genetic elements in the germline. suggestion that miRNA-mediated repression 2006; Eulalio et al., 2008a). Moreover, tethering Such mobile elements can randomly integrate occurs at early steps of translation. Using of GW182 to a target mRNA represses into the genome and it is important that such density-gradient fractions, it was shown translation of the mRNA independently of events are repressed in the germline. Recently, it that mRNAs repressed by miRNAs do not Drosophila AGO1, demonstrating that GW182 has been found that Piwi proteins are methylated sediment in polysome fractions, but shift to is an important effector protein functioning by the arginine methyltransferase PRMT5 and the free messenger ribonucleoprotein pool. downstream of Ago proteins (Behm-Ansmant possibly by other methyltransferases. These Furthermore, it was shown that miRNA- et al., 2006). A recent study in Krebs-2 mouse post-translational modifications enable Piwi mediated translational repression could only ascite cell extract (Fabian et al., 2009) and an proteins to interact with Tudor-domain- target mRNAs containing a functional m7G cap earlier study in Drosophila (Behm-Ansmant containing proteins (TDRDs) (Kirino et al., (Filipowicz et al., 2008; Pillai et al., 2007). It has et al., 2006) found that CAF1, a component of 2009; Reuter et al., 2009; Vagin et al., 2009). therefore been suggested that miRNA-guided the CCR-NOT deadenylase complex that is Not much is known about the function of TDRD translational repression is based on preventing required for the removal of the poly(A) tail of proteins in . It has been suggested the circularization of the mRNA that is needed mRNAs, is at least partially responsible that TDRD1 contributes to pre-pachytene for stimulation of translation. Contradicting this for miRNA-mediated deadenylation of target piRNA biogenesis and is important for silencing model are studies in which mRNA transcripts mRNAs. Furthermore, poly(A)-binding protein repetitive elements. lacking a poly(A) tail can still be targeted by (PABP), which interacts with the poly(A) tail of In addition to piRNAs, the endo-siRNA miRNA-mediated repression (Carthew and mRNAs and the eukaryotic translation initiation pathway also contributes to transposon Sontheimer, 2009; Filipowicz et al., 2008; Pillai factor 4G (eIF4G) subunit of the cap-binding repression (Okamura and Lai, 2008). In the male et al., 2007). In contrast to the translation complex that circularizes the mRNA during germline, mutations in components of the piRNA

Journal of Cell Science initiation model of miRNA function, evidence translational initiation, also interacts with pathway have a severe effect on fertility, whereas of miRNA-mediated effects during later GW182 proteins (Beilharz et al., 2009; Fabian this pathway appears to be dispensable in the stages of translation has been reported. It has et al., 2009). Consistently, it has been proposed female germline of mammals, which is enriched been suggested that miRNA causes mRNAs to that GW182 proteins might compete with eIF4G in endo-siRNAs (Tam et al., 2008; Watanabe prematurely dissociate from or ‘drop off’ for PABP binding and therefore prevent mRNA et al., 2008). It might therefore be possible that, in . Furthermore, an independent model circularization. Uncircularized mRNA might the germline, the piRNA pathway and the endo- proposes that the nascent polypeptide chain not be able to initiate translation efficiently and siRNA pathway cooperate with each other in translated from the mRNA target is degraded co- protein expression is reduced (Beilharz et al., transposon repression. translationally (Carthew and Sontheimer, 2009; 2009; Fabian et al., 2009; Zekri et al., 2009). Although thousands of individual piRNA Filipowicz et al., 2008). In summary, Ago proteins are highly sequences have been identified, they derive In addition to their capacity to mediate specialized small-RNA-binding proteins. Small from discrete genomic clusters (Aravin and translational repression, it has been RNAs guide Ago proteins to complementary Hannon, 2008). Drosophila Piwi subfamily demonstrated that miRNAs can guide the target mRNAs, where Ago proteins act together members PIWI and AUB bind to piRNAs that destabilization of target mRNAs that contain with protein binding partners to interfere with are antisense to transposon RNAs, whereas the imperfectly complementary target sites (Bagga translation or induce deadenylation of target third Piwi protein in flies, AGO3, typically et al., 2005; Behm-Ansmant et al., 2006; mRNAs. binds the sense strands. AUB- and PIWI- Giraldez et al., 2006; Wu and Belasco, 2005). associated piRNAs preferentially carry a uracil The mechanism of miRNA-mediated mRNA Piwi proteins, piRNA function and at their 5 end, whereas most AGO3-associated decay requires proteins of the mRNA- transposon silencing piRNAs have an adenine at nucleotide 10. With degradation machinery and depends on a In Drosophila, PIWI, Aubergine (AUB) the discovery that the first ten nucleotides of member of the Ago protein family and and AGO3 constitute the Piwi subfamily. antisense piRNAs are often complementary to a member of the GW182 protein family Mutations in the PIWI protein lead to defects the sense piRNAs, the ‘ping-pong’ model has (Behm-Ansmant et al., 2006; Eulalio et al., in oogenesis and depletion of germline cells, been proposed (Kim et al., 2009). In this model, 2008b; Jakymiw et al., 2005; Liu et al., 2005; whereas AUB mutations disrupt gametogenesis. AUB or PIWI proteins that are associated with Meister et al., 2005). GW182 is characterized by In mice, all Piwi proteins are important for antisense piRNAs cleave sense the presence of glycine and tryptophan repeats . The discovery of piRNAs, the transcripts, thereby creating the 5 end of sense (GW repeats) and localizes to processing bodies small RNA partners to which Piwi proteins bind, piRNAs that then associate with AGO3. AGO3 1822 Journal of Cell Science 123 (11)

Wohlschlegel, J. A., Sachidanandam, R. et al. (2008). An subsequently cleaves antisense retrotransposon work is needed. Further supporting the idea that endogenous small interfering RNA pathway in Drosophila. transcripts, generating the 5 end of antisense small-RNA pathways are functional in the Nature 453, 798-802. piRNAs that subsequently bind to PIWI. It is not nucleus, it has also recently been reported that Drinnenberg, I. A., Weinberg, D. E., Xie, K. T., Mower, J. P., Wolfe, K. H., Fink, G. R. and Bartel, D. P. (2009). known how the 3 end of the piRNA is Ago proteins localize to the nucleus (Weinmann RNAi in budding yeast. Science 326, 544-550. generated. However, it is tempting to speculate et al., 2009). However, the mechanistic details Ender, C., Krek, A., Friedlander, M. R., Beitzinger, M., that exonucleases or interact with of small-RNA function in the nucleus of Weinmann, L., Chen, W., Pfeffer, S., Rajewsky, N. and Meister, G. (2008). A human snoRNA with microRNA-like Piwi proteins, and either trim or cleave the RNA mammalian cells remain unclear. functions. Mol. Cell 32, 519-528. to generate the correct piRNA 3 end. Through Eulalio, A., Behm-Ansmant, I. and Izaurralde, E. (2007). this continuous cycle, piRNAs are amplified and Perspectives P bodies: at the crossroads of post-transcriptional pathways. Nat. Rev. Mol. Cell Biol. 8, 9-22. retrotransposon silencing can be maintained. Although many aspects of Argonaute function Eulalio, A., Huntzinger, E. and Izaurralde, E. (2008a). However, the mechanism by which piRNA have been characterized, there are still many Getting to the root of miRNA-mediated gene silencing. Cell biogenesis is initiated is not understood. It is 132, 9-14. open questions. Why do only some Argonaute Eulalio, A., Huntzinger, E. and Izaurralde, E. (2008b). known that AUB and PIWI are maternally proteins possess activity, despite GW182 interaction with Argonaute is essential for miRNA- inherited (Brennecke et al., 2008). It is possible containing crucial conserved amino acids? Two mediated translational repression and mRNA decay. Nat. that the maternally contributed piRNAs that are Struct. Mol. Biol. 15, 346-353. recent publications have reported that Fabian, M. R., Mathonnet, G., Sundermeier, T., Mathys, associated with AUB and PIWI act as primary Argonaute proteins might have different H., Zipprich, J. T., Svitkin, Y. V., Rivas, F., Jinek, M., piRNAs and initiate the ping-pong cycle in the cleavage preferences: whereas only human Wohlschlegel, J., Doudna, J. A. et al. (2009). Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP- . The ping-pong model found in AGO2 cleaves complementary target mRNAs dependent deadenylation. Mol. Cell 35, 868-880. Drosophila might also be applicable to mouse through an RNAi-like mechanism, both AGO1 Filipowicz, W., Bhattacharyya, S. N. and Sonenberg, N. piRNAs and the mouse Piwi proteins MILI and and AGO2 can cleave the passenger strand of an (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, MIWI2. siRNA duplex (Steiner et al., 2009; Wang, B. 102-114. et al., 2009). However, further work will be Ghildiyal, M., Seitz, H., Horwich, M. D., Li, C., Du, T., Argonaute proteins in the nucleus Lee, S., Xu, J., Kittler, E. L., Zapp, M. L., Weng, Z. et necessary to confirm these initial observations. al. (2008). Endogenous siRNAs derived from transposons In addition to their role in small-RNA-mediated It is also becoming increasingly apparent that and mRNAs in Drosophila somatic cells. Science 320, 1077- silencing at the mRNA level in the cytoplasm, Argonaute proteins can be post-translationally 1081. Argonaute proteins are also thought to function Giraldez, A. J., Mishima, Y., Rihel, J., Grocock, R. J., modified: modifications such as hydroxylation, Van Dongen, S., Inoue, K., Enright, A. J. and Schier, A. in the nucleus at the transcriptional level. phosphorylation and ubiquitylation influence F. (2006). Zebrafish MiR-430 promotes deadenylation and Transcriptional gene silencing was first Argonaute stability and function in mammals clearance of maternal mRNAs. Science 312, 75-79. discovered in plants, in which small RNAs Jakymiw, A., Lian, S., Eystathioy, T., Li, S., Satoh, M., (Qi et al., 2008; Rybak et al., 2009; Zeng et al., Hamel, J. C., Fritzler, M. J. and Chan, E. K. (2005). derived from transgenes and viral RNAs were 2008). However, Argonaute modifications have Disruption of GW bodies impairs mammalian RNA found to guide methylation of homologous not yet been analyzed systematically. Future interference. Nat. Cell Biol. 7, 1267-1274. DNA sequences. 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