Erratum The role of GW/P-bodies in RNA processing and silencing Andrew Jakymiw, Kaleb M. Pauley, Songqing Li, Keigo Ikeda, Shangli Lian, Theophany Eystathioy, Minoru Satoh, Marvin J. Fritzler and Edward K. L. Chan

Journal of Cell Science 120, 1702 (2007) doi:10.1242/jcs.03452 There was an error published in J. Cell Sci. 120, 1317-1323.

We apologise for two errors that occurred in the online and pdf versions of this article. The printed version is correct.

On p. 1317, in the Summary, the sentence ‘Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA.’ appeared twice. The correct version of the summary is shown below.

Summary GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that RNA-induced silencing complexes may be activated within these cytoplasmic centers. Work over the past few years has significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components that spatially regulate mRNA turnover in various biological processes. Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate in successive steps into larger ribonucleoprotein structures.

On p. 1319, right column, first paragraph, the word order of the penultimate sentence was incorrect and should read:

In particular, studies in Drosophila indicate that GW182 interacts with Ago1 and promotes miRNA-mediated degradation of a subset of mRNA targets (Behm-Ansmant et al., 2006). Commentary 1317 The role of GW/P-bodies in RNA processing and silencing

Andrew Jakymiw1, Kaleb M. Pauley1, Songqing Li1, Keigo Ikeda1, Shangli Lian1, Theophany Eystathioy2, Minoru Satoh3, Marvin J. Fritzler2 and Edward K. L. Chan1,* 1Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA 2Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada 3Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA *Author for correspondence (e-mail: [email protected])

Accepted 21 February 2007 Journal of Cell Science 120, 1317-1323 Published by The Company of Biologists 2007 doi:10.1242/jcs.03429

Summary GW bodies, also known as mammalian P-bodies, are significantly increased our understanding of the biology of cytoplasmic foci involved in the post-transcriptional GW bodies, revealing that they are specialized cell regulation of eukaryotic gene expression. Recently, GW components that spatially regulate mRNA turnover in bodies have been linked to RNA interference and various biological processes. The formation of GW bodies demonstrated to be important for short-interfering-RNA- appears to depend on both specific protein factors and and microRNA-mediated mRNA decay and translational RNA, in particular, microRNA. Here, we propose a repression. Evidence indicates that both passenger and working model for GW body assembly in terms of its guide strands of short-interfering RNA duplexes can relationship to RNA interference. In this process, one or localize to GW bodies, thereby indicating that RNA- more heteromeric protein complexes accumulate in induced silencing complexes may be activated within these successive steps into larger ribonucleoprotein structures. cytoplasmic centers. Formation of GW bodies appears to depend on both specific protein factors and RNA, in Key words: GW bodies, P-bodies, RNA interference, mRNA particular, microRNA. Work over the past few years has degradation, microRNA, short-interfering RNA

Introduction mRNAs (for reviews, see Filipowicz et al., 2005; Meister and

Journal of Cell Science The control of mRNA stability plays key roles in both the post- Tuschl, 2004; Rana, 2007; Sen and Blau, 2006; Valencia- transcriptional regulation of eukaryotic gene expression Sanchez et al., 2006). The discovery that RNAi effector proteins (Keene and Lager, 2005; Wilusz and Wilusz, 2004) and mRNA (Jakymiw et al., 2005; Liu et al., 2005a; Liu et al., 2005b; Sen quality control (Fasken and Corbett, 2005). The latter involves and Blau, 2005) and small RNAs localize to GWBs (Jakymiw et the recognition and rapid degradation of aberrant mRNAs and al., 2005; Pauley et al., 2006; Pillai et al., 2005), and that GWB takes place when translation termination occurs too early assembly appears to be required for the proper functioning of the (nonsense-mediated decay) or fails to occur (non-stop decay) RNAi pathway (Jakymiw et al., 2005; Liu et al., 2005a; Meister (Fasken and Corbett, 2005) or when translation elongation et al., 2005), suggests that these foci are specifically involved in stalls (no-go decay) (Doma and Parker, 2006). In eukaryotes, short-interfering RNA (siRNA)- and microRNA (miRNA)- mRNA turnover is regulated by two major mechanisms. One mediated mRNA degradation and/or translational repression†. involves the multisubunit exosome, where transcripts are degraded by 3Ј-to-5Ј exonucleases (for a review, see van Hoof †siRNAs are small RNAs of ~21 nucleotides in length and are derived from the progressive cleavage of long, perfectly complementary double-stranded RNAs (dsRNAs) by an and Parker, 1999). The second mechanism involves RNase-III-type endonuclease, Dicer. They can originate from long dsRNAs transiently cytoplasmic compartments termed GW bodies (GWBs), which introduced into cells by transfection or stably expressed hairpin-containing dsRNA Ј Ј precursors derived from DNA constructs. They assemble into an RNA-protein complex spatially control mRNA turnover by the 5 -to-3 mRNA decay known as the RNA-induced silencing complex (RISC or siRISC), which includes machinery. These discrete cytoplasmic foci, also called Dcp- Argonaute 2 (Ago2), a key component of RNAi that possesses endonuclease activity. containing bodies or processing (P)-bodies, constitute sites of RISC then targets and cleaves perfectly complementary mRNAs, generating 5Ј and 3Ј fragments, which are subsequently degraded. MiRNAs are similar in size to siRNAs but mRNA degradation, storage and translational repression originate from hairpin-containing precursors encoded by the genome. These non-coding (Brengues et al., 2005; Coller and Parker, 2005; Cougot et al., precursors of miRNAs have double-stranded regions with imperfect complementarity and 2004; Eystathioy et al., 2002; Eystathioy et al., 2003; Sheth are sequentially processed by RNase-III-type enzymes Drosha and Dicer into mature miRNAs. The mature miRNAs then assemble into an RNA-protein complex referred to and Parker, 2003; Van Dijk et al., 2002). Recently, they have as the miRNA ribonucleoprotein complex (miRNP or miRISC), which is structurally also been shown to function in RNA interference (RNAi) similar to RISC and contains at least one Argonaute protein. Multiple copies of miRNPs are directed to the 3Ј-UTR of certain mRNAs that have imperfect complementarity to the (Jakymiw et al., 2005; Liu et al., 2005a; Liu et al., 2005b; bound miRNAs. In plants, miRNAs cleave their regulated target mRNA, whereas in Meister et al., 2005; Pillai et al., 2005; Sen and Blau, 2005). mammals miRNAs promote translational repression of the targeted mRNA. Until recently, RNAi is a post-transcriptional silencing mechanism in which this was considered the major distinction between siRISC and miRISC in mammalian cells. However, new evidence suggests that miRNAs can also regulate mRNA degradation small double-stranded RNA molecules induce sequence-specific similarly to siRNAs (Bagga et al., 2005; Jing et al., 2005; Yekta et al., 2004), thus blurring degradation and/or translational repression of homologous the distinction between the two small-RNA-mediated silencing complexes. 1318 Journal of Cell Science 120 (8)

GWBs are enriched in mRNA decay factors and pools of Segal et al., 2006). Furthermore, there are functional stored messenger ribonucleoproteins (mRNPs) (Bruno and differences between GWBs and yeast P-bodies in terms of their Wilkinson, 2006; Sheth and Parker, 2006). Moreover, they are responses to stress and cell growth (Schneider et al., 2006). For dynamic structures, whose size and number appear to depend example, P-bodies increase in size and number during growth on specific intracellular processes. For instance, GWBs vary in limitation, increased cell density, and stress (Teixeira et al., size and number throughout the cell cycle, the largest of which 2005), whereas GWBs increase in size and number in are observed in late S and G2 phase (Yang et al., 2004). proliferating cells (Yang et al., 2004) and dynamically interact Furthermore, stress (Kedersha et al., 2005; Teixeira et al., with stress granules‡ in stressed mammalian cells (Kedersha et 2005), cell proliferation (Yang et al., 2004), blocking mRNA al., 2005). Therefore, one needs to be cautious when decay (Andrei et al., 2005; Cougot et al., 2004; Sheth and generalizing about these structures because differences do Parker, 2003) and inhibition of translational initiation exist, not only between species but even within a population of (Brengues et al., 2005; Sheth and Parker, 2003; Teixeira et al., GWBs in a single mammalian cell (Fig. 1). Interestingly, a 2005) all increase the size and number of GWBs. Conversely, recent report similarly found that some GW/P-bodies that blocking transcription, deadenylation of mRNAs or contain Mex-3B, a newly described class of human RNA- translational elongation decreases the size and number of binding protein that localizes to GW/P-bodies, are also devoid GWBs (Cougot et al., 2004; Sheth and Parker, 2003). Recent of Dcp1 (Buchet-Poyau et al., 2007). The function of a studies have highlighted the importance of these structures in particular GW/P-body may therefore depend on its the regulation of mRNA turnover and provided insights into organization or composition. their involvement in RNA silencing. Here, we review the current understanding of GWB function, focusing on its recent GW body function links to RNAi, and discuss the role of these cytoplasmic foci mRNA degradation in RNA-induced silencing complex (RISC) activation. We also The decay of mRNA in GW/P-bodies is thought to occur by examine the requirements for GWB formation and disassembly a 5Ј-to-3Ј exonucleolytic process, which first requires the and propose a working model for their genesis. removal of the 3Ј-poly(adenosine) [poly(A)] tail – deadenylation. Subsequently, the 5Ј mRNA cap is irreversibly GWBs uncovered removed by a decapping complex and the body of the mRNA GWBs were first identified and characterized in studies using is degraded by the 5Ј-to-3Ј exonuclease Xrn1 (for a review, an autoimmune serum from a human patient with motor and see Coller and Parker, 2004). Evidence also suggests that sensory neuropathy (Eystathioy et al., 2002). They were named GW/P-bodies can regulate translation (Bhattacharyya et al., as such because they harbor the mRNA-binding protein 2006; Coller and Parker, 2005; Ferraiuolo et al., 2005; Pillai GW182. GW182 is characterized by glycine (G) and et al., 2005) and store mRNAs (Bhattacharyya et al., 2006; tryptophan (W) repeat-rich domains and a canonical RNA- Brengues et al., 2005; Pillai et al., 2005). Moreover, GW/P- recognition motif and has been demonstrated to associate with bodies also appear to be involved in other mRNA decay a specific subset of transcripts in HeLa cells (Eystathioy et al., pathways, such as nonsense mediated decay (NMD), AU-rich

Journal of Cell Science 2002). The finding that a set of mammalian proteins involved element (ARE)-mediated decay (AMD) and stress-induced in mRNA degradation – Dcp1/2, Xrn1 and LSm1-7 – localize decay (SID). to similar prominent cytoplasmic foci (Bashkirov et al., 1997; NMD is a surveillance mechanism that removes aberrant Eystathioy et al., 2003; Ingelfinger et al., 2002; Van Dijk et al., mRNA transcripts containing premature termination codons. 2002), and that GWBs are active sites of mRNA decay in Several factors involved in NMD, including UPF1-3, SMG5 human cells (Cougot et al., 2004), indicated that GWBs are and SMG7, as well as reporter mRNAs harboring nonsense involved in the spatial regulation of mRNA turnover, and more mutations have been found to localize to GW/P-bodies specifically, a selective 5Ј-to-3Ј mRNA degradation pathway. (Fukuhara et al., 2005; Sheth and Parker, 2006; Unterholzner Concurrent studies identified GWB-related structures referred and Izaurralde, 2004), which links these foci and the NMD to as P-bodies in budding yeast, and these were found to play process. GWBs have also been linked to AMD, a decay process a role in mRNA decapping and 5Ј-to-3Ј decay (Sheth and involving messages containing AREs in the 3Ј-untranslated Parker, 2003). In particular, Sheth and Parker demonstrated region (UTR). In particular, depletion of three GWB proteins, that insertion of a poly(G) tract into mRNAs, which blocks 5Ј- Xrn1, LSm1 or 4E-T, has been demonstrated to inhibit AMD to-3Ј exonuclease activity, causes accumulation of decay (Ferraiuolo et al., 2005; Stoecklin et al., 2006). Moreover, intermediates within P-bodies, suggesting that the mRNA evidence also indicates that the ARE-binding protein TTP, decay process is associated with them (Sheth and Parker, which is known to destabilize ARE-containing mRNAs, 2003). localizes to GWBs (Kedersha et al., 2005) and interacts with These bodies are thought to be conserved in budding yeast and activates the decapping complex (Fenger-Gron et al., 2005; and mammals because both the yeast P-bodies and mammalian Lykke-Andersen and Wagner, 2005). Finally, the observation GWBs contain activators of decapping and decapping enzymes that the size and number of P-bodies increases during stress in (Cougot et al., 2004; Eystathioy et al., 2003; Ingelfinger et al., budding yeast cells (Teixeira et al., 2005) and that GWBs can 2002; Lykke-Andersen, 2002; Segal et al., 2006; Sheth and be induced to form and interact with stress granules by specific Parker, 2003; Van Dijk et al., 2002). Note, however, that GWBs stresses in mammalian cells demonstrates their importance in differ from yeast P-bodies in that they contain translation SID (Kedersha et al., 2005). However, because budding yeast initiation factors and other proteins that have no yeast

counterparts, such as factors involved in RNAi [e.g. GW182 ‡Stress granules are large RNP particles that store non-translating mRNAs when cells are and Argonaute (Ago) proteins] (Anderson and Kedersha, 2006; exposed to environmental stresses, but are absent in budding yeast cells. GW/P-bodies in RNA processing and silencing 1319

Fig. 1. GW bodies (GWBs) are heterogeneous structures with obvious differences in protein composition. (Top) HEp-2 cells stained with human anti-GWB serum (a prototype serum often used as a GWB marker and known to contain antibodies to GW182, hAgo2 and Ge-1, but not Dcp1; green) and rabbit anti-Dcp1 antibody (often used as a marker for P-bodies; red) demonstrate that not all GWBs contain Dcp1. (Bottom) HEp-2 cells stained with mouse anti-hAgo2 monoclonal (green) and rabbit anti-RCK/p54 (red) polyclonal antibodies demonstrate that not all foci containing RCK/p54 contain hAgo2. Conversely, not all foci that stain for hAgo2 contain RCK/p54. Arrows, GWBs that do not contain both protein factors. Arrowheads, GWBs that do contain both protein factors. Nuclei (blue) were counterstained with DAPI. Bar, 10 ␮m.

cells lack stress granules, the mechanism of SID probably 2005; Liu et al., 2005a; Liu et al., 2005b). Moreover, differs from that in higher eukaryotes. depletion of GW182, which disrupts GWBs (Yang et al., In mammalian cells, GWBs and stress granules have been 2004), perturbs both siRNA- and miRNA-mediated reported to be compositionally and morphologically distinct repression (Jakymiw et al., 2005; Liu et al., 2005a). Dominant structures (Cougot et al., 2004; Kedersha et al., 2005); interfering GW182 and hAgo2 mutants also disrupt GWB however, evidence indicates that they are functionally and formation and similarly inhibit RNA silencing (Jakymiw et spatially linked (Kedersha et al., 2005). Interestingly, both al., 2005). The impairment in RNAi appears to depend on entities share an assortment of proteins (e.g. CPEB1, Rck/p54, blocking the localization of hAgo2 to GWBs (Jakymiw et al., FAST, Xrn1, eIF4 and TTP) and a single class of reporter 2005). Indeed, hAgo2 constructs containing point mutations mRNA has been observed within both GWBs and stress that prevent siRNA binding and localization to GWBs do not granules (Kedersha et al., 2005; Wilczynska et al., 2005). repress target reporter mRNA, despite being tethered to the Moreover, overexpression of TTP and CPEB1, a translational target (Liu et al., 2005a). Additional evidence for the regulator, induces the fusion of stress granules and GWBs, involvement of GW182 and GWB in RNAi comes from

Journal of Cell Science which suggests that these proteins regulate the dynamic studies of the Drosophila melanogaster ortholog and interaction between these two structures (Kedersha et al., 2005; Caenorhabditis elegans functional analog (Ding et al., 2005; Wilczynska et al., 2005). One model is that stress granules Rehwinkel et al., 2005; Schneider et al., 2006). In particular, serve as mRNA triage sites where transcripts are sorted for studies in Drosophila indicate that GW182 interacts with storage, re-initiation of translation, or degradation, and that Ago1 and promotes degradation of a subset of miRNA- mRNAs targeted for decay are exported from stress granules mediated-decay mRNA targets (Behm-Ansmant et al., 2006). to GWBs (Kedersha et al., 2005). Regardless of the GW182 may thus function as a molecular scaffold that mechanistic differences between yeast and higher eukaryotes, bridges components of the miRNA pathway with mRNA it is evident that GW/P-bodies have multiple functions, the decay enzymes (Behm-Ansmant et al., 2006). ultimate goal being mRNA decay and/or storage. It is therefore A point of contention is whether GW182 and GWBs are not surprising that these foci comprise multiple factors and that important for both slicer-dependent mechanisms (i.e. siRNA- their specific structural composition and organization probably mediated RNAi) and miRNA-mediated repression or only the determines their mode of action. latter. Work from our laboratory has demonstrated that transfected fluorophore-labeled siRNAs associate with GW182 RNAi protein complexes and localize to GWBs and that GW182 and Several groups recently demonstrated a link between GWBs GWBs play an important role in slicer-mediated functions and RNAi (Jakymiw et al., 2005; Liu et al., 2005a; Liu et al., (Jakymiw et al., 2005). Moreover, Liu et al. found that 2005b; Pillai et al., 2005; Sen and Blau, 2005). In particular, suppression of GW182 similarly impairs, albeit not as the Ago family of proteins, Ago1-4, which are components of effectively, the ability of an siRNA to silence its target by RISC, the key effector complex of RNAi, were found to be mRNA cleavage (Liu et al., 2005a). In addition, silencing of concentrated in GWBs (Jakymiw et al., 2005; Liu et al., 2005a; the GWB protein TNRC6B, a GW182 paralog, inhibits Liu et al., 2005b; Sen and Blau, 2005). Moreover, GWBs also cleavage of an mRNA reporter gene containing a target site appeared to be sites involved in miRNA-mediated repression perfectly complementary to an endogenous miRNA (Meister of targeted mRNAs (Liu et al., 2005b). et al., 2005). TNRC6B thus appears to be important for slicer- In addition, we and others demonstrated that hAgo2, the dependent mechanisms and subsequent mRNA degradation. catalytic engine of RNA silencing, associates with several Other work has indicated a greater role for GW182 and components of GWBs, including GW182 (Jakymiw et al., GWBs in miRNA-mediated translational repression by 1320 Journal of Cell Science 120 (8)

Table 1. Suppression of specific GWB factors demonstrates their interdependence on accumulation within and for assembly of GWBs Target* Cell function GWB foci† GWB markers examined References GW182 RNAi – LSm4, hAgo2, hDcp1a, hDcp2 (Jakymiw et al., 2005; Liu et al., 2005a; Yang et al., 2004) Xrn1 5Ј-to-3Ј exonuclease + hDcp2 (Andrei et al., 2005; Cougot et al., 2004) Ccr4 Deadenylation – LSm1, RCK/p54, eIF4E, eIF4E-T (Andrei et al., 2005) LSm1 Decapping – Ccr4, RCK/p54, eIF4E, eIF4E-T (Andrei et al., 2005; Chu and Rana, 2006) LSm4 Decapping – GW182, hDcp1a (Kedersha et al., 2005) hDcp2 Decapping + LSm1, RCK/p54, eIF4E, eIF4E-T, Ge-1 (Andrei et al., 2005; Yu et al., 2005) RCK/p54 Decapping; translation – hAgo2, LSm1, Ccr4, eIF4E, eIF4E-T (Andrei et al., 2005; Chu and Rana, 2006) control; RNAi eIF4E-T Translation control – Ccr4, RCK/p54, eIF4E, LSm1, hDcp1 (Andrei et al., 2005; Ferraiuolo et al., 2005) RAP55 Translation control – hDcp1a (Tanaka et al., 2006; Yang et al., 2006) Ge-1/Hedls Decapping – hDcp1a, hDcp2 (Yu et al., 2005) miRNA‡ RNAi – GW182, hAgo2, hDcp1a (Pauley et al., 2006) mRNA§ Translation – hDcp1a, LSm1, RCK/p54, (Andrei et al., 2005; Cougot et al., 2004) eIF4E, eIF4E-T

GWB, GW body; miRNA, microRNA; RNAi, RNA interference; siRNA, short-interfering RNA; *proteins silenced through siRNA-mediated gene-specific knockdown; †detected using fluorescence microscopy; ‡miRNA suppression was induced indirectly through siRNA-mediated knockdown of Drosha or DGCR8; §accumulation within GWBs inhibited using cycloheximide or actinomycin D.

demonstrating, using various reporter systems, that depletion strand into RISC may occur within GWBs. Docking of of GW182 impairs miRNA function (Chu and Rana, 2006; Liu siRNA/miRNA duplexes into RISC and its subsequent et al., 2005a; Rehwinkel et al., 2005). Furthermore, depletion activation could therefore be early events in GWB formation. of GW182 has also been demonstrated to result in alterations Interestingly, in fission yeast cells, a Dicer ortholog localizes of mRNA expression profiles very similar to those seen in cells to structures resembling GWBs (Carmichael et al., 2006), depleted of the Drosophila miRNA effector Ago1 and not the which suggests that GWB formation may take place even siRNA effector Ago2, which suggests that GW182 functions before RISC activation. in the miRNA pathway (Behm-Ansmant et al., 2006). Whether the above disparities are because of the use of different reporter GW body formation and structure systems (i.e. exogenously introduced versus endogenous Immunogold electron microscopy of GWBs identifies reporter systems), species variations or GW182 redundancy cytoplasmic electron-dense structures that are 100-300 nm in (three paralogs have been identified in humans) will require diameter and which lack a membrane (Eystathioy et al., 2002; further study. Interestingly, in the study implicating GW182 in Yang et al., 2004). Closer inspection reveals that they comprise

Journal of Cell Science slicer-mediated function, we used siRNAs with a fluorophore 8-10 nm strands or fibrils (Yang et al., 2004). Currently, the conjugated to the 5Ј-end of the guide strand (Jakymiw et al., mechanism of GWB formation is not well understood. It 2005). The fluorophore might therefore have interfered with remains unclear whether GWBs form de novo or whether the siRNA activity, making it behave more like an miRNA by mRNAs and their associated proteins are targeted to pre- producing imperfect base-pairing between siRNA and target. existing structures. Furthermore, we do not know whether Regardless, the studies collectively demonstrate a role for GWB components are targeted to GWBs independently or as GW182 and GWBs in RNAi. More work will be needed to part of larger complexes that form higher-order structures that determine whether GWBs represent converging sites for can be visualized by conventional light microscopy. RNAi- siRISC and/or miRISC. mediated depletion of specific GWB factors in human cells has MiRNAs have similarly been identified within GWBs and demonstrated an interdependence of each of the proteins for demonstrated to associate with GW182 protein complexes their accumulation in GWBs (see Table 1). GWBs may thus (Pauley et al., 2006; Pillai et al., 2005). The identification of form by the assembly of one or more heteromeric protein siRNA and/or miRNA within GWBs suggests that RISC complexes on mRNAs that can amass into larger mRNP activation, activity and/or recycling may occur within GWBs. structures. The finding that specific enzymes involved in Are GWBs sites of RISC activation and activity? Evidence decapping and subsequent 5Ј-to-3Ј degradation are dispensable supporting this possibility includes the observation that Ago2, for GWB formation (Andrei et al., 2005) – unlike factors a known GWB component, is directly involved in passenger- involved at earlier stages of mRNA decay (e.g. mRNA 3Ј-end strand cleavage of double-stranded siRNAs during RISC trimming) – indicates that earlier stages are more crucial for assembly (Matranga et al., 2005; Rand et al., 2005). Also, GWB assembly. following transient transfection of HeLa cells with a Fig. 2 shows our working model, in which several factors, fluorophore-labeled passenger-strand siRNA duplex that has including RISC, initially interact with the target mRNA to form no endogenous mRNA target, the siRNA localizes to GWBs – a specific RNP structure dependent on the type of decay or similarly to a fluorophore-labeled-guide-strand siRNA duplex storage process that will occur (e.g. siRNA-mediated decay that has an endogenous mRNA target (Jakymiw et al., 2005). versus miRNA-mediated translational repression). This results This suggests that the passenger strand and the guide strand in the recruitment of other protein complexes, which depend localize to GWBs independently of the mRNA target and that on the composition of the initial RNP; so their final passenger-strand cleavage and incorporation of the antisense- composition or structural organization promotes the proper GW/P-bodies in RNA processing and silencing 1321

Fig. 2. A model linking RNAi and GW body (GWB) assembly and function. RNAi activity is triggered by siRNA/miRNA duplexes, which are first processed from either double-stranded RNA (dsRNA) or precursor microRNA (pre-miRNA), respectively, by the RNase-III-type endonuclease Dicer. These duplexes are then incorporated into RISC, where the passenger strand (black) is either cleaved and degraded or Journal of Cell Science removed by the bypass mechanism (Matranga et al., 2005). This activation process and assembly of the guide strand (red) into RISC is thought to initiate early stages of GWB formation. Subsequent targeting by RISC results in further recruitment of one or more heteromeric protein complexes (which could include GW182 and RCK/p54) on the mRNA, which forms a specific RNP structure that causes post-transcriptional inhibition of gene expression (through siRNA-mediated cleavage or miRNA-mediated translational repression, depending on the degree of complementarity between the guide-strand RNA and its target mRNA). The targeted mRNA is eventually degraded by further recruitment of 5Ј- to-3Ј mRNA decay factors, which include the deadenylase Ccr4, decapping factors (LSm1-7 ring, Dcp complex) and the 5Ј-to-3Ј exonuclease Xrn1. The accumulation of RNA decay factors during degradation could be responsible for the size increases in GWBs, which makes them visible by conventional light microscopy. ORF, open reading frame.

decay mechanism and/or storage of the mRNA. Such a model integral component of GW/P-bodies (Andrei et al., 2005; would explain why disruption of GWBs by depletion of other Cougot et al., 2004; Eystathioy et al., 2002; Teixeira et al., GWB components, such as LSm1 or RCK/p54, does not 2005). Furthermore, silencing of Drosha or its required partner necessarily translate into impaired RISC activity (Chu and protein, DGCR8, which together comprise the microprocessor Rana, 2006), whereas silencing of GW182 does (Jakymiw et complex and are responsible for processing long nuclear al., 2005). One possibility is that GW182 exists in a smaller primary miRNA (pri-miRNA) transcripts to ~70-nucleotide RNP complex with RISC components (e.g. hAgo2) that is hairpin precursor miRNA (pre-miRNA), depletes cells of undetectable by fluorescence microscopy and supersedes the mature miRNA and causes the disappearance of GWBs (Pauley requirement of LSm1 and RCK/p54 for initial target et al., 2006). This suggests that miRNAs are required for the recognition and endonucleolytic cleavage or translation formation of GWBs. Although the possibility that the effect is repression. Moreover, not all silencing of GWB factors impairs indirect cannot be ruled out, this seems unlikely because formation of GWBs equivalently. Interestingly, Andrei et al. siRNA transfected into Drosha-deficient cells can serve as a have demonstrated that silencing of RCK/p54 has a less surrogate for miRNA and drive the reappearance of GWBs. pronounced effect on the disassembly of GWBs compared with The dependence of GWBs on miRNA means that many of other factors (Andrei et al., 2005). the functions attributed to GWBs similarly may depend on The integrity of GWBs also appears to depend on RNA. miRNAs in mammalian cells. Processes such as translational Studies in yeast and mammals indicate that mRNA is an repression, mRNA decay and storage depend on miRNAs. 1322 Journal of Cell Science 120 (8)

Furthermore, miRNAs are involved in ARE-mediated nuclear RNAi pathways are interrelated and the relationship degradation events (Jing et al., 2005). All of these processes between GWBs and nuclear RNAi are currently unclear. are associated with GWBs. The primary function of GWBs Biochemical evidence suggests that these processes are linked might therefore be to provide a microenvironment for miRNA- by a common requirement for Ago proteins; however, mRNA interactions that lead to translational inhibition and/or immunofluorescence studies using a newly developed mouse mRNA degradation. Whether NMD processes share this monoclonal antibody specific for hAgo2 show no evidence of requirement for miRNAs requires further study. Nevertheless, nuclear localization of this endogenous protein (Ikeda et al., if this hypothesis holds, a more appropriate name for these foci 2006), which is consistent with our earlier work using in mammalian cells may be miRNA-induced bodies (miRBs). polyclonal autoimmune sera (Jakymiw et al., 2006). Regardless, the recent findings that GWBs appear to function Conclusions and perspectives in siRNA/miRNA-mediated forms of post-transcriptional Recent cell biology and biochemical findings have regulation, the apparent requirement for GWBs in Drosophila significantly enhanced our understanding of the spatial (Schneider et al., 2006) and C. elegans (Ding et al., 2005) regulation of mRNA decay and/or storage within eukaryotic development, and the knowledge that small RNAs regulate cells, and it is now evident that dynamic cytoplasmic foci, many cellular activities (Ambros, 2004), including GW/P-bodies, are crucial for these processes. The discovery of differentiation, stem cell division, and apoptosis, underline the a functional link between GWBs and RNAi has been importance of GWBs for many biological processes. particularly instrumental in allowing us to decipher the complexities of how small RNAs and their cognate proteins are We thank Jens Lykke-Andersen (University of Colorado) and Tom involved in post-transcriptional gene regulation (for reviews, Hobman (University of Alberta) for their generosity in providing see Engels and Hutvagner, 2006; Eulalio et al., 2007; Jackson valuable antibody reagents. We apologize to colleagues whose and Standart, 2007; Pillai et al., 2007; Rana, 2007). interesting work could not be cited owing to space limitations. This work was supported in part by the National Institutes of Health Grant Our understanding of the cell biology of RNAi and its AI47859 and the Canadian Institutes for Health Research Grant MOP- relationship to GWBs is still limited, however. During the 38034 and the Canadian Breast Cancer Research Foundation Grant course of writing this review, several articles were published 16992. demonstrating that a large fraction of miRNAs and Ago proteins reside in the cytoplasm (Leung et al., 2006; Maroney et al., 2006; Nottrott et al., 2006), in particular on mRNAs References being actively translated by polyribosomes (Maroney et al., Ambros, V. (2004). The functions of animal microRNAs. Nature 431, 350-355. Anderson, P. and Kedersha, N. (2006). RNA granules. J. Cell Biol. 172, 803-808. 2006; Nottrott et al., 2006). Furthermore, inhibiting translation Andrei, M. A., Ingelfinger, D., Heintzmann, R., Achsel, T., Rivera-Pomar, R. and initiation or inducing stress has been shown to result in Luhrmann, R. (2005). A role for eIF4E and eIF4E-transporter in targeting mRNPs to localization of miRNA and Ago to stress granules (Leung et mammalian processing bodies. RNA 11, 717-727. Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R. and Pasquinelli, al., 2006). Data from these studies suggest that miRNPs first A. E. (2005). Regulation by let-7 and lin-4 miRNAs results in target mRNA associate with and suppress actively translating mRNAs in the degradation. Cell 122, 553-563. Bashkirov, V. I., Scherthan, H., Solinger, J. A., Buerstedde, J. M. and Heyer, W. D. Journal of Cell Science cytoplasm prior to completely dropping off ribosomes. Upon (1997). A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 miRNA-mediated repression, these mRNA are then believed to tetraplex substrates. J. Cell Biol. 136, 761-773. be targeted to either stress granules for storage or sorted and Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P. and Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT shuttled to GWBs for decay. Given the close relationship deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885-1898. between GWBs and stress granules, this would not be Berezhna, S. Y., Supekova, L., Supek, F., Schultz, P. G. and Deniz, A. A. (2006). surprising; however, one needs to be cautious in interpreting siRNA in human cells selectively localizes to target RNA sites. Proc. Natl. Acad. Sci. USA 103, 7682-7687. the stress granule data because stress granules are generally Bhattacharyya, S. N., Habermacher, R., Martine, U., Closs, E. I. and Filipowicz, W. observed only during a stress response, whereas GWBs are (2006). Relief of microRNA-mediated translational repression in human cells subjected present continuously (Teixeira et al., 2005). Alternatively, to stress. Cell 125, 1111-1124. Brengues, M., Teixeira, D. and Parker, R. (2005). Movement of eukaryotic mRNAs inhibition of actively translating polysomes on mRNAs by between polysomes and cytoplasmic processing bodies. Science 310, 486-489. miRISCs might directly trigger the recruitment of RNP Bruno, I. and Wilkinson, M. F. (2006). P-bodies react to stress and nonsense. Cell 125, 1036-1038. complexes involved in either early stages of GWB formation Buchet-Poyau, K., Courchet, J., Hir, H. L., Seraphin, B., Scoazec, J. Y., Duret, L., and/or GWB targeting. Much more work will be needed if we Domon-Dell, C., Freund, J. N. and Billaud, M. (2007). Identification and are to completely understand the interactions between GWBs characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies. Nucleic Acids Res. and other mRNP structures and the significance of their In press. heterogeneous composition. Carmichael, J. B., Stoica, C., Parker, H., McCaffery, J. M., Simmonds, A. J. and Finally, although numerous studies have demonstrated that Hobman, T. C. (2006). RNA interference effector proteins localize to mobile cytoplasmic puncta in Schizosaccharomyces pombe. Traffic 7, 1032-1044. RNAi occurs in the cytoplasm of a cell, a growing number of Chu, C. Y. and Rana, T. M. (2006). Translation repression in human cells by microRNA- reports suggest that it occurs within the nucleus as well. In induced gene silencing requires RCK/p54. PLoS Biol. 4, e210. Coller, J. and Parker, R. (2004). Eukaryotic mRNA decapping. Annu. Rev. Biochem. 73, particular, recent studies in human cells have implicated the 861-890. RNAi effector proteins hAgo1 and hAgo2 in the post- Coller, J. and Parker, R. (2005). General translational repression by activators of mRNA transcriptional modulation of gene expression in the nucleus decapping. Cell 122, 875-886. Cougot, N., Babajko, S. and Seraphin, B. (2004). Cytoplasmic foci are sites of mRNA (Robb et al., 2005) and transcriptional silencing (Janowski et decay in human cells. J. Cell Biol. 165, 31-40. al., 2006; Kim et al., 2006). Furthermore, transfection of Ding, L., Spencer, A., Morita, K. and Han, M. (2005). The developmental timing siRNAs targeting small nuclear RNAs (e.g. 7SK and U6 RNA) regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol. Cell 19, 437-447. leads to their translocation into the nucleus and silencing of the Doma, M. K. and Parker, R. (2006). Endonucleolytic cleavage of eukaryotic mRNAs target genes (Berezhna et al., 2006). How the cytoplasmic and with stalls in translation elongation. Nature 440, 561-564. GW/P-bodies in RNA processing and silencing 1323

Engels, B. M. and Hutvagner, G. (2006). Principles and effects of microRNA-mediated Meister, G. and Tuschl, T. (2004). Mechanisms of gene silencing by double-stranded post-transcriptional gene regulation. Oncogene 25, 6163-6169. RNA. Nature 431, 343-349. Eulalio, A., Behm-Ansmant, I. and Izaurralde, E. (2007). P bodies: at the crossroads Meister, G., Landthaler, M., Peters, L., Chen, P. Y., Urlaub, H., Luhrmann, R. of post-transcriptional pathways. Nat. Rev. Mol. Cell Biol. 8, 9-22. and Tuschl, T. (2005). Identification of novel argonaute-associated proteins. Curr. Eystathioy, T., Chan, E. K., Tenenbaum, S. A., Keene, J. D., Griffith, K. and Fritzler, Biol. 15, 2149-2155. M. J. (2002). A phosphorylated cytoplasmic autoantigen, GW182, associates with a Nottrott, S., Simard, M. J. and Richter, J. D. (2006). Human let-7a miRNA blocks unique population of human mRNAs within novel cytoplasmic speckles. Mol. Biol. protein production on actively translating polyribosomes. Nat. Struct. Mol. Biol. 13, Cell 13, 1338-1351. 1108-1114. Eystathioy, T., Jakymiw, A., Chan, E. K., Seraphin, B., Cougot, N. and Fritzler, M. Pauley, K. M., Eystathioy, T., Jakymiw, A., Hamel, J. C., Fritzler, M. J. and Chan, J. (2003). The GW182 protein colocalizes with mRNA degradation associated proteins E. K. (2006). Formation of GW bodies is a consequence of microRNA genesis. hDcp1 and hLSm4 in cytoplasmic GW bodies. RNA 9, 1171-1173. EMBO Rep. 7, 904-910. Fasken, M. B. and Corbett, A. H. (2005). Process or perish: quality control in mRNA Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N., Basyuk, E., biogenesis. Nat. Struct. Mol. Biol. 12, 482-488. Bertrand, E. and Filipowicz, W. (2005). Inhibition of translational initiation by Fenger-Gron, M., Fillman, C., Norrild, B. and Lykke-Andersen, J. (2005). Multiple Let-7 MicroRNA in human cells. Science 309, 1573-1576. processing body factors and the ARE binding protein TTP activate mRNA decapping. Pillai, R. S., Bhattacharyya, S. N. and Filipowicz, W. (2007). Repression of protein Mol. Cell 20, 905-915. synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 17, 118-126. Ferraiuolo, M. A., Basak, S., Dostie, J., Murray, E. L., Schoenberg, D. R. and Rana, T. M. (2007). Illuminating the silence: understanding the structure and function Sonenberg, N. (2005). A role for the eIF4E-binding protein 4E-T in P-body formation of small RNAs. Nat. Rev. Mol. Cell Biol. 8, 23-36. and mRNA decay. J. Cell Biol. 170, 913-924. Rand, T. A., Petersen, S., Du, F. and Wang, X. (2005). Argonaute2 cleaves the anti- Filipowicz, W., Jaskiewicz, L., Kolb, F. A. and Pillai, R. S. (2005). Post-transcriptional guide strand of siRNA during RISC activation. Cell 123, 621-629. gene silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol. 15, 331-341. Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. and Izaurralde, E. (2005). A crucial Fukuhara, N., Ebert, J., Unterholzner, L., Lindner, D., Izaurralde, E. and Conti, E. role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene (2005). SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay silencing. RNA 11, 1640-1647. pathway. Mol. Cell 17, 537-547. Robb, G. B., Brown, K. M., Khurana, J. and Rana, T. M. (2005). Specific and potent Ikeda, K., Satoh, M., Pauley, K. M., Fritzler, M. J., Reeves, W. H. and Chan, E. K. RNAi in the nucleus of human cells. Nat. Struct. Mol. Biol. 12, 133-137. (2006). Detection of the argonaute protein Ago2 and microRNAs in the RNA induced Schneider, M. D., Najand, N., Chaker, S., Pare, J. M., Haskins, J., Hughes, S. C., silencing complex (RISC) using a monoclonal antibody. J. Immunol. Methods 317, 38- Hobman, T. C., Locke, J. and Simmonds, A. J. (2006). Gawky is a component of 44. cytoplasmic mRNA processing bodies required for early Drosophila development. Ingelfinger, D., Arndt-Jovin, D. J., Luhrmann, R. and Achsel, T. (2002). The human J. Cell Biol. 174, 349-358. LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in Segal, S. P., Dunckley, T. and Parker, R. (2006). Sbp1p affects translational distinct cytoplasmic foci. RNA 8, 1489-1501. repression and decapping in Saccharomyces cerevisiae. Mol. Cell. Biol. 26, 5120- Jackson, R. J. and Standart, N. (2007). How do microRNAs regulate gene expression? 5130. Sci. STKE 2007, re1. Sen, G. L. and Blau, H. M. (2005). Argonaute 2/RISC resides in sites of mammalian Jakymiw, A., Lian, S., Eystathioy, T., Li, S., Satoh, M., Hamel, J. C., Fritzler, M. J. mRNA decay known as cytoplasmic bodies. Nat. Cell Biol. 7, 633-636. and Chan, E. K. (2005). Disruption of GW bodies impairs mammalian RNA Sen, G. L. and Blau, H. M. (2006). A brief history of RNAi: the silence of the genes. interference. Nat. Cell Biol. 7, 1267-1274. FASEB J. 20, 1293-1299. Jakymiw, A., Ikeda, K., Fritzler, M. J., Reeves, W. H., Satoh, M. and Chan, E. K. Sheth, U. and Parker, R. (2003). Decapping and decay of messenger RNA occur in (2006). Autoimmune targeting of key components of RNA interference. Arthritis Res. cytoplasmic processing bodies. Science 300, 805-808. Ther. 8, R87. Sheth, U. and Parker, R. (2006). Targeting of aberrant mRNAs to cytoplasmic Janowski, B. A., Huffman, K. E., Schwartz, J. C., Ram, R., Nordsell, R., Shames, D. processing bodies. Cell 125, 1095-1109. S., Minna, J. D. and Corey, D. R. (2006). Involvement of AGO1 and AGO2 in Stoecklin, G., Mayo, T. and Anderson, P. (2006). ARE-mRNA degradation requires mammalian transcriptional silencing. Nat. Struct. Mol. Biol. 13, 787-792. the 5Ј-3Ј decay pathway. EMBO Rep. 7, 72-77. Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova, F., Tanaka, K. J., Ogawa, K., Takagi, M., Imamoto, N., Matsumoto, K. and Lin, S. C., Gram, H. and Han, J. (2005). Involvement of microRNA in AU-rich Tsujimoto, M. (2006). RAP55, a cytoplasmic mRNP component, represses element-mediated mRNA instability. Cell 120, 623-634. translation in Xenopus oocytes. J. Biol. Chem. 281, 40096-40106. Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fitzler, M. Teixeira, D., Sheth, U., Valencia-Sanchez, M. A., Brengues, M. and Parker, R. J., Scheuner, D., Kaufman, R. J., Golan, D. E. and Anderson, P. (2005). Stress (2005). Processing bodies require RNA for assembly and contain nontranslating

Journal of Cell Science granules and processing bodies are dynamically linked sites of mRNP remodeling. J. mRNAs. RNA 11, 371-382. Cell Biol. 169, 871-884. Unterholzner, L. and Izaurralde, E. (2004). SMG7 acts as a molecular link between Keene, J. D. and Lager, P. J. (2005). Post-transcriptional operons and regulons co- mRNA surveillance and mRNA decay. Mol. Cell 16, 587-596. ordinating gene expression. Chromosome Res. 13, 327-337. Valencia-Sanchez, M. A., Liu, J., Hannon, G. J. and Parker, R. (2006). Control of Kim, D. H., Villeneuve, L. M., Morris, K. V. and Rossi, J. J. (2006). Argonaute-1 directs translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515- siRNA-mediated transcriptional gene silencing in human cells. Nat. Struct. Mol. Biol. 524. 13, 793-797. Van Dijk, E., Cougot, N., Meyer, S., Babajko, S., Wahle, E. and Seraphin, B. Leung, A. K., Calabrese, J. M. and Sharp, P. A. (2006). Quantitative analysis of (2002). Human Dcp2: a catalytically active mRNA decapping enzyme located in Argonaute protein reveals microRNA-dependent localization to stress granules. Proc. specific cytoplasmic structures. EMBO J. 21, 6915-6924. Natl. Acad. Sci. USA 103, 18125-18130. van Hoof, A. and Parker, R. (1999). The exosome: a proteasome for RNA? Cell 99, Liu, J., Rivas, F. V., Wohlschlegel, J., Yates, J. R., III, Parker, R. and Hannon, G. J. 347-350. (2005a). A role for the P-body component GW182 in microRNA function. Nat. Cell Wilczynska, A., Aigueperse, C., Kress, M., Dautry, F. and Weil, D. (2005). The Biol. 7, 1261-1266. translational regulator CPEB1 provides a link between dcp1 bodies and stress Liu, J., Valencia-Sanchez, M. A., Hannon, G. J. and Parker, R. (2005b). MicroRNA- granules. J. Cell Sci. 118, 981-992. dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7, Wilusz, C. J. and Wilusz, J. (2004). Bringing the role of mRNA decay in the control 719-723. of gene expression into focus. Trends Genet. 20, 491-497. Lykke-Andersen, J. (2002). Identification of a human decapping complex associated Yang, W. H., Yu, J. H., Gulick, T., Bloch, K. D. and Bloch, D. B. (2006). RNA- with hUpf proteins in nonsense-mediated decay. Mol. Cell. Biol. 22, 8114-8121. associated protein 55 (RAP55) localizes to mRNA processing bodies and stress Lykke-Andersen, J. and Wagner, E. (2005). Recruitment and activation of mRNA decay granules. RNA 12, 547-554. enzymes by two ARE-mediated decay activation domains in the proteins TTP and Yang, Z., Jakymiw, A., Wood, M. R., Eystathioy, T., Rubin, R. L., Fritzler, M. J. BRF-1. Genes Dev. 19, 351-361. and Chan, E. K. (2004). GW182 is critical for the stability of GW bodies expressed Maroney, P. A., Yu, Y., Fisher, J. and Nilsen, T. W. (2006). Evidence that microRNAs during the cell cycle and cell proliferation. J. Cell Sci. 117, 5567-5578. are associated with translating messenger RNAs in human cells. Nat. Struct. Mol. Biol. Yekta, S., Shih, I. H. and Bartel, D. P. (2004). MicroRNA-directed cleavage of 13, 1102-1107. HOXB8 mRNA. Science 304, 594-596. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. and Zamore, P. D. (2005). Passenger- Yu, J. H., Yang, W. H., Gulick, T., Bloch, K. D. and Bloch, D. B. (2005). Ge-1 is a strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme central component of the mammalian cytoplasmic mRNA processing body. RNA 11, complexes. Cell 123, 607-620. 1795-1802.