Tom20 Mediates Localization of mRNAs to Mitochondria in a Translation-Dependent Manner

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20Δ puf3Δ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20Δ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.mRNA localization to distinct cellular compartments is important for the efficiency and specificity of the translation process. Synthesis of proteins at their sites of action may decrease the likelihood of ectopic protein expression and facilitate assembly of large multiprotein complexes. Two general modes for mRNA localization are known. The first, which is common for endoplasmic reticulum (ER)-associated mRNAs, necessitates translation of a short region of the protein (the signal peptide). The signal is recognized by the signal recognition particle as it emerges from the ribosome exit tunnel, and the complex that includes the mRNA, ribosome, and signal recognition particle is targeted to the ER (18). As an outcome of this process, mRNAs that encode proteins destined for the ER and the secretory pathway are associated with this compartment (7). The second mode for mRNA localization occurs prior to translation and in many cases prevents initiation of protein synthesis. Sequences or structural elements of the mRNA are bound by RNA-binding proteins, and these interact with transport factors, which direct the mRNA to its destination (5, 35, 42). Genome-wide studies indicate that localization by either mode is a broad phenomenon that encompasses many mRNAs and various cellular destinations (6, 21, 32, 38). Interestingly, we along with others have recently shown that noncoding regions may also be involved in localization of ER-associated mRNAs (1, 26, 38), demonstrating that these two modes are not mutually exclusive.Most of the mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Various in vitro and in vivo assays led to the widely accepted notion that import may occur posttranslationally, i.e., after the protein is fully synthesized in the cytosol (33). However, mounting evidence also supports a cotranslational import of proteins into the mitochondria. Specifically, polysomes were shown to be associated with the mitochondrial surface, and these translated a distinct set of proteins (12, 19, 20). Moreover, isolated mitochondria are associated with many different mRNAs that encode mitochondrial proteins (28, 46). Elements from both the coding region (the mitochondrial targeting signal [MTS]) and the 3′ untranslated region (UTR) were shown to be important for targeting of some of these mRNAs (4, 29). One model for localization suggests association of the nascent peptide chain (specifically, the N-terminal MTS) with receptors on the mitochondria, coupled to cotranslational insertion of the protein (24). As an outcome of this cotranslational mechanism, polysomal mRNAs become associated with the mitochondria, analogously to what is observed in the ER. However, experimental support for this hypothesis is currently lacking.Recently, Saint-Georges et al. (41) have shown a role for Puf3p in localization of many mRNAs to the mitochondria of Saccharomyces cerevisiae. Puf3p is associated with the mitochondria outer membrane (11), and its role is mediated through interaction with UTRs. This may suggest a translation-independent mode of action. Intriguingly, however, most Puf3 targets appeared to be mislocalized also after treatment with the translation inhibitor cycloheximide (CHX), suggesting that an active translation process is required for their asymmetric localization (41). Moreover, a large number of mRNAs that are not Puf3 targets appeared to be affected from treatments with the translation inhibitors puromycin and cycloheximide (41), further supporting the existence of an additional, translation-dependent mode of mRNA targeting to the mitochondria.The translocase of the mitochondrial outer membrane (TOM complex) is a multiprotein machinery which mediates the import of the vast majority of proteins into the mitochondria (36, 39). Its core protein (Tom40) forms a β-barrel structure and serves as the main component of the import pore. Tom20 is a peripheral component of the TOM complex that functions as a primary receptor for mitochondrial precursor proteins (15). It was hypothesized that protein receptors interact with the incoming polypeptide while it is translated, and this leads to a local increase in mRNA concentration (24). An open question is whether the TOM complex, through Tom20, interacts with polypeptides while they are translated and thereby leads to higher local concentrations of mRNAs near the mitochondria. To address this issue, we analyzed the effects of TOM20 deletion on mRNA association with mitochondrial fractions and the role of the MTS on mRNA localization. We also tested the interactions between Tom20 and Puf3. We found that Tom20 is involved in mitochondrial association of many mRNAs by a process that requires the MTS. Tom20 deletion affects the localization of Puf3p, and a strain with deletions of both Tom20 and Puf3 exhibits a growth defect under conditions that require mitochondrial optimal function.     

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m⁷G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m⁷G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m⁷G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m⁷G cap at the 5′ end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G''s interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5′ to 3′ until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5′ untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5′ UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5′ UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5′ UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with cap-dependent translation, the cap and the 5′ UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3, 8, 17, 26, 36, 47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17, 28, 39, 55, 56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5′ end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5′ UTR. In metazoa, the m⁷G cap is replaced with a trimethylguanosine (TMG) cap (m^2,2,7GpppN) (27, 30, 46, 49). In nematodes, ∼70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing metazoa effectively recognizes TMG-capped trans-spliced mRNAs, what role the SL sequence plays in translation initiation, and how the conserved translation initiation machinery has adapted to effectively translate trans-spliced mRNAs.Previous work has shown that efficient translation of TMG-capped messages in nematodes requires the SL sequence (22 nt) immediately downstream of the cap (5, 25, 29). In the current studies, we sought to understand the manner in which the SL enhanced the translation of TMG-capped mRNAs. Using a cell-free nematode in vitro translation system, we carried out mutational analyses that define the specific sequences in the SL that are required and sufficient for efficient translation of TMG-capped mRNAs. These analyses led to the discovery of a small, discrete stem-loop immediately adjacent to the TMG cap in trans-spliced messages required for efficient translation. Notably, the sequences involved in the base pairing of the stem are highly conserved in alternative SL sequences found in nematodes. We further show that the nematode eIF4E/G complex plays a major role in facilitating the SL enhancement of TMG-capped mRNA that likely occurs after the initial cap-binding step. The results demonstrate the importance of specific enhancing elements in the 5′ UTR and adaptation in the eIF4F complex necessary for optimal cap-dependent translation.     

Evidence for Translational Regulation by the Herpes Simplex Virus Virion Host Shutoff Protein

The herpes simplex virus (HSV) virion host shutoff protein (vhs) encoded by gene UL41 is an mRNA-specific RNase that triggers accelerated degradation of host and viral mRNAs in infected cells. We report here that vhs is also able to modulate reporter gene expression without greatly altering the levels of the target mRNA in transient-transfection assays conducted in HeLa cells. We monitored the effects of vhs on a panel of bicistronic reporter constructs bearing a variety of internal ribosome entry sites (IRESs) located between two test cistrons. As expected, vhs inhibited the expression of the 5′ cistrons of all of these constructs; however, the response of the 3′ cistron varied with the IRES: expression driven from the wild-type EMCV IRES was strongly suppressed, while expression controlled by a mutant EMCV IRES and the cellular ApaF1, BiP, and DAP5 IRES elements was strongly activated. In addition, several HSV type 1 (HSV-1) 5′ untranslated region (5′ UTR) sequences also served as positive vhs response elements in this assay. IRES activation was also observed in 293 and HepG2 cells, but no such response was observed in Vero cells. Mutational analysis has yet to uncouple the ability of vhs to activate 3′ cistron expression from its shutoff activity. Remarkably, repression of 5′ cistron expression could be observed under conditions where the levels of the reporter RNA were not correspondingly reduced. These data provide strong evidence that vhs can modulate gene expression at the level of translation and that it is able to activate cap-independent translation through specific cis-acting elements.The virion host shutoff protein (vhs) encoded by herpes simplex virus (HSV) gene UL41 is an endoribonuclease that is packaged into the tegument of mature HSV virions. Once delivered into the cytoplasm of newly infected cells, vhs triggers shutoff of host protein synthesis, disruption of preexisting polysomes, and degradation of host mRNAs (reviewed in reference 62). The vhs-dependent shutoff system destabilizes many cellular and viral mRNAs (36, 46, 67). The rapid decline in host mRNA levels presumably helps viral mRNAs gain access to the cellular translational apparatus. In addition, the relatively short half-lives of viral mRNAs contribute to the sharp transitions between the successive phases of viral protein synthesis by tightly coupling changes in the rates of synthesis of viral mRNAs to altered mRNA levels (46). These effects enhance virus replication and may account for the modest reduction in virus yield displayed by vhs mutants in cultured Vero cells (55, 61).vhs also plays a critical role in HSV pathogenesis: vhs mutants are severely impaired for replication in the corneas and central nervous systems of mice and cannot efficiently establish or reactivate from latency (63, 65, 66). Mounting evidence indicates that this attenuation stems at least in part from an impaired ability to disarm elements of the innate and adaptive host immune responses (reviewed in reference 62). For example, vhs suppresses certain innate cellular antiviral responses, including production of proinflammatory cytokines and chemokines (68); dampens the type I interferon system (11, 45, 49, 78); and blocks activation of dendritic cells (58). Moreover, vhs mutants display enhanced virulence in knockout mice lacking type I interferon (IFN) receptors (37, 45) or Stat1 (48) and are hypersensitive to the antiviral effects of IFN in some cells in tissue culture (11, 49, 68). Thus, vhs is arguably a bona fide virulence factor.vhs present in extracts of HSV virions or purified from bacteria has nonspecific RNase activity capable of degrading all RNA substrates (15, 70, 71, 79). However, vhs is highly selective in vivo, targeting mRNAs and sparing other cytoplasmic RNAs (36, 46). In vivo and in mammalian whole-cell extracts, vhs-induced decay of at least some mRNAs initiates near regions of translation initiation and proceeds in an overall 5′-to-3′ direction (12, 13, 29, 52). Moreover, vhs binds to the translation initiation factors eIF4H, eIF4B, and eIF4A II, all components of the cap recognition factor eIF4F (10, 16, 17). Thus, it has been proposed that vhs selectively targets actively translated mRNAs through interactions with eIF4F components (17). Consistent with this hypothesis, recent data document that eIF4H is required for vhs activity in vivo (59).A previous report from this laboratory documented that the internal ribosome entry sites (IRESs) of the picornaviruses poliovirus and encephalomyocarditis virus (EMCV) strongly target vhs-induced RNA cleavage events to sequences immediately 3′ to the IRES in an in vitro translation system derived from rabbit reticulocyte lysates (RRL) (13). IRES elements are highly structured RNA sequences that are able to direct cap-independent translational initiation (reviewed in references 21, 25, 30, and 64). In the case of the poliovirus and EMCV elements, this is achieved by directly recruiting the eIF4F scaffolding protein eIF4G, thus bypassing the requirement for the cap-binding eIF4F subunit, eIF4E (reviewed in reference 30). Based on these data, we suggested that vhs is strongly targeted to the picornavirus IRES elements via interactions with eIF4 factors.A growing number of cellular mRNAs have been proposed to bear IRES elements in their 5′ untranslated regions (5′ UTRs). These include many that are involved in cellular stress responses, apoptosis, and cell cycle progression (24, 64, 74). Given the striking ability of picornavirus IRES elements to target vhs RNase activity in vitro, we asked whether viral and cellular IRES elements are able to modify the susceptibility of mRNAs to vhs in vivo. During the course of preliminary experiments designed to test this hypothesis, we unexpectedly discovered that vhs is able to strongly activate gene expression controlled by some cellular IRES elements and HSV 5′ UTR sequences in in vivo bicistronic reporter assays. These observations are the subject of the present report.     

Control of Cell Survival and Proliferation by Mammalian Eukaryotic Initiation Factor 4B

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5′ untranslated region (5′UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5′UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.Targeting the translation initiation pathway is emerging as a potential therapy for inhibiting cancer cell growth (35, 38). Ribosome recruitment to the 5′ ends of eukaryotic mRNAs proceeds via translation initiation mechanisms that are dependent either on the 5′ cap structure (m⁷GpppN, where N is any nucleotide) or an internal ribosome entry site (IRES). The majority of translation initiation events in eukaryotes are mediated through cap-dependent translation whereby the 40S ribosomal subunit is recruited to the vicinity of the mRNA 5′ cap structure by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of eIF4E (the cap-binding subunit), eIF4A (an RNA helicase), and eIF4G (a large scaffolding protein for eIF4E, eIF4A, and other initiation factors). Once assembled at the 5′ cap, the 40S ribosomal subunit in association with several initiation factors scans the 5′ untranslated region (5′UTR) of the mRNA until it encounters a start codon in a favorable context, followed by polypeptide synthesis (37).Early in vitro studies have shown that the initiation factor eIF4B acts to potentiate ribosome recruitment to the mRNA (3, 45). eIF4B stimulates translation of both capped and uncapped mRNAs in vitro (1, 36). This function is exerted through stimulation of the helicase activity of eIF4A (43), possibly through direct interactions with eIF4A (44) or with mRNA, the ribosome-associated eIF3, and 18S rRNA (28, 29, 44). Thus, eIF4B is thought to form auxiliary bridges between the mRNA and the 40S ribosomal subunit. Toeprinting studies using mammalian eIF4B underscored its importance in the assembly of the 48S initiation complex, especially on mRNAs harboring secondary structures in the 5′UTRs (11).In vivo studies of eIF4B are limited. Ectopic expression of eIF4B in cultured Drosophila melanogaster cells and in developing eye imaginal discs stimulated cell proliferation (16). Enhanced cell proliferation is most likely mediated by increased translation of a subset of mRNAs, since knockdown of Drosophila eIF4B by RNA interference (RNAi) caused a modest reduction in global translation but compromised the survival of insect cells grown under low serum conditions (16). Studies of eIF4B in mammalian cells yielded contradictory results. Transient overexpression of eIF4B stimulated translation initiation in a phosphorylation-dependent manner in some cells (18, 49) while inhibiting translation in others (30, 31, 41). These differences might be attributed to disparate levels of eIF4B overexpression.To address the physiological role of eIF4B in mRNA translation in the cell, RNAi knockdown of eIF4B was used here. We demonstrate that eIF4B is required for optimal translation. Importantly, the translation of mRNAs bearing structured 5′UTRs, such as the cell cycle regulators Cdc25C, c-myc, and ODC (ornithine decarboxylase), and the antiapoptotic factors Bcl-2 and XIAP (X-linked inhibitor of apoptosis), was reduced as a result of eIF4B silencing by RNAi. Furthermore, eIF4B silencing promoted caspase-dependent apoptosis. Thus, we show that mammalian eIF4B is required for cell proliferation and survival, whereby it acts by regulating the translation of a functionally related subset of mRNAs.     

The Silencing Domain of GW182 Interacts with PABPC1 To Promote Translational Repression and Degradation of MicroRNA Targets and Is Required for Target Release

GW182 family proteins are essential in animal cells for microRNA (miRNA)-mediated gene silencing, yet the molecular mechanism that allows GW182 to promote translational repression and mRNA decay remains largely unknown. Previous studies showed that while the GW182 N-terminal domain interacts with Argonaute proteins, translational repression and degradation of miRNA targets are promoted by a bipartite silencing domain comprising the GW182 middle and C-terminal regions. Here we show that the GW182 C-terminal region is required for GW182 to release silenced mRNPs; moreover, GW182 dissociates from miRNA targets at a step of silencing downstream of deadenylation, indicating that GW182 is required to initiate but not to maintain silencing. In addition, we show that the GW182 bipartite silencing domain competes with eukaryotic initiation factor 4G for binding to PABPC1. The GW182-PABPC1 interaction is also required for miRNA target degradation; accordingly, we observed that PABPC1 associates with components of the CCR4-NOT deadenylase complex. Finally, we show that PABPC1 overexpression suppresses the silencing of miRNA targets. We propose a model in which the GW182 silencing domain promotes translational repression, at least in part, by interfering with mRNA circularization and also recruits the deadenylase complex through the interaction with PABPC1.In multicellular eukaryotes, the regulation of gene expression by microRNAs (miRNAs) is critical for biological processes as diverse as cell differentiation and proliferation, apoptosis, metabolism, and development (4). To exert a regulatory function, miRNAs associate with Argonaute proteins to form RNA-induced silencing complexes, which repress translation and trigger the degradation of target mRNAs (4, 10, 16). The extent to which translational repression and degradation contribute to silencing depends on the specific target-miRNA combination; some targets are regulated predominantly at the translational level, whereas others can be regulated mainly at the mRNA level (3). A large-scale proteomic analysis performed in parallel with measurements of mRNA levels showed that for the vast majority of miRNA targets, silencing correlates with changes at both the protein and mRNA levels (1, 27).In animal cells, the degradation of miRNA targets is initiated by deadenylation and decapping, which are followed by the exonucleolytic decay of the mRNA body (2, 3, 9, 11, 12, 17, 19, 24, 30, 31). miRNA-dependent mRNA degradation requires a variety of proteins: an Argonaute and a GW182 protein, the CCR4-NOT deadenylase complex, the decapping enzyme DCP2, and several decapping activators including DCP1, Ge-1, HPat, EDC3, and Me31B (also known as RCK/p54) (3, 6, 9, 12, 19). Several studies previously demonstrated that miRNAs trigger deadenylation and decapping even when the mRNA target is not translated (9, 12, 19, 24, 30, 31), indicating that mRNA decay is not merely a consequence of a primary effect of miRNAs on translation but rather is an independent mechanism by which miRNAs silence gene expression.Although how miRNAs trigger mRNA degradation is well established, the mechanisms driving the inhibition of translation are unclear. Multiple mechanisms have been proposed: the displacement of eukaryotic initiation factor 4E (eIF4E) from the mRNA cap structure, interference with the function of the eIF4F complex, a block of 60S ribosomal subunit joining, or an inhibition of translation elongation (4, 10, 16). Regardless of the precise mechanism, the translational repression of miRNA targets also requires GW182 family proteins (11, 13).GW182 proteins are essential components of the miRNA pathway in animal cells, as their depletion suppresses miRNA-mediated gene silencing (reviewed in references 8 and 13). Recent studies have revealed that the silencing activity of these proteins resides predominantly in a bipartite silencing domain containing the middle and C-terminal regions (14, 22, 33). The precise molecular function of the GW182 silencing domain is not fully understood, yet it is known that the domain is not required for GW182 proteins to interact with Argonaute proteins or to localize to P bodies (3, 14, 22). Furthermore, when the silencing domains of GW182 proteins are artificially tethered to mRNAs, their expression is silenced; therefore, tethering bypasses the requirement for Argonaute proteins and miRNAs (5, 22, 33). These observations suggest that the silencing domains of GW182 proteins exhibit intrinsic silencing activity and therefore likely play a role at the effector step of silencing (13, 14, 22, 33).Here we investigate what role the Drosophila melanogaster GW182 silencing domain plays in the miRNA pathway. Overall, our results reveal that the very C-terminal region of this domain is required for the release of GW182 from silenced mRNPs. Indeed, we unexpectedly found that we could detect D. melanogaster GW182 bound to miRNA targets only in cells depleted of components of the deadenylase complex. These results suggest that GW182 dissociates from Argonaute-1 (AGO1) and miRNA targets at a step of silencing downstream of deadenylation. In contrast, GW182 mutants lacking the C-terminal region remain stably bound to miRNA targets, even in wild-type cells, indicating that this region plays a role in the dissociation of GW182 from effector complexes. We further show that the bipartite silencing domain of GW182 interacts with PABPC1 and interferes with the binding of PABPC1 to eIF4G. The interaction of GW182 with PABPC1 is also required for the degradation of miRNA targets, most likely because the interaction facilitates the recruitment of the CCR4-NOT deadenylase complex. Accordingly, overexpressing PABPC1 suppresses the silencing of miRNA targets. Our findings uncover an unexpected role for PABPC1 in the miRNA pathway.