The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2–PAN3 and CCR4–NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.     

miRISC and the CCR4–NOT complex silence mRNA targets independently of 43S ribosomal scanning

miRNAs associate with Argonaute (AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4–NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4–NOT complex in Drosophila melanogaster. Here, we show that miRNAs, AGOs, GW182, the CCR4–NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning‐independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and miRNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning.     

Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo

RNA-regulatory factors bound to 3′ UTRs control translation and stability. Repression often is associated with poly(A) removal. The deadenylase CAF1 is a core component of the CCR4–NOT complex. Our prior studies established that CAF1 represses translation independent of deadenylation. We sought the mechanism of its deadenylation-independent repression in Xenopus oocytes. Our data reveal a chain of interacting proteins that links CAF1 to CCR4–NOT and to Xp54 and 4E-T. Association of CAF1 with NOT1, the major subunit of CCR4–NOT, is required for repression by CAF1 tethered to a reporter mRNA. Affinity purification-mass spectrometry and coimmunoprecipitation revealed that at least five members of the CCR4–NOT complex were recruited by CAF1. The recruitment of these proteins required NOT1, as did the ability of tethered CAF1 to repress translation. In turn, NOT1 was needed to recruit Xp54 and 4E-T. We examined the role of 4E-T in repression using mutations that disrupted either eIF4E-dependent or -independent mechanisms. Expression of a 4E-T truncation that still bound eIF4E alleviated repression by tethered CAF1, NOT1, and Xp54. In contrast, a mutant 4E-T that failed to bind eIF4E did not. Repression of global translation was affected only by the eIF4E-dependent mechanism. Reporters bearing IRES elements revealed that repression via tethered CAF1 and Xp54 is cap- and eIF4E-independent, but requires one or more of eIF4A, eIF4B, and eIF4G. We propose that RNA-binding proteins, and perhaps miRNAs, repress translation through an analogous chain of interactions that begin with the 3′ UTR-bound repressor and end with the noncanonical activity of 4E-T.     

The Caenorhabditis elegans GW182 protein AIN-1 interacts with PAB-1 and subunits of the PAN2-PAN3 and CCR4-NOT deadenylase complexes

GW182 family proteins are essential for miRNA-mediated gene silencing in animal cells. They are recruited to miRNA targets via interactions with Argonaute proteins and then promote translational repression and degradation of the miRNA targets. The human and Drosophila melanogaster GW182 proteins share a similar domain organization and interact with PABPC1 as well as with subunits of the PAN2-PAN3 and CCR4-NOT deadenylase complexes. The homologous proteins in Caenorhabditis elegans, AIN-1 and AIN-2, lack most of the domains present in the vertebrate and insect proteins, raising the question as to how AIN-1 and AIN-2 contribute to silencing. Here, we show that both AIN-1 and AIN-2 interact with Argonaute proteins through GW repeats in the middle region of the AIN proteins. However, only AIN-1 interacts with C. elegans and D. melanogaster PABPC1, PAN3, NOT1 and NOT2, suggesting that AIN-1 and AIN-2 are functionally distinct. Our findings reveal a surprising evolutionary plasticity of the GW182 protein interaction network and demonstrate that binding to PABPC1 and deadenylase complexes has been maintained throughout evolution, highlighting the significance of these interactions for silencing.     

HPat a Decapping Activator Interacting with the miRNA Effector Complex

Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in Drosophila cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.     

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.     

The C-terminal domains of human TNRC6A,TNRC6B,and TNRC6C silence bound transcripts independently of Argonaute proteins

Proteins of the GW182 family are essential components of the miRNA pathway in animal cells. Vertebrate genomes encode three GW182 paralogs (TNRC6A, TNRC6B, and TNRC6C), which may be functionally redundant. Here, we show that the N-terminal GW-repeat-containing regions of all three TNRC6s interact with the four human Argonaute proteins (AGO1–AGO4). We also show that TNRC6A, TNRC6B, and TNRC6C silence the expression of bound mRNAs. This activity is mediated by their C-terminal silencing domains, and thus, is independent of the interaction with AGO1–AGO4. Silencing by TNRC6A, TNRC6B, and TNRC6C is effected by changes in protein expression and mRNA stability that can, in part, be attributed to deadenylation. Our findings indicate that TNRC6A, TNRC6B, and TNRC6C are recruited to miRNA targets through an interaction between their N-terminal domain and an Argonaute protein; the TNRC6s then promote translational repression and/or degradation of miRNA targets through a C-terminal silencing domain.     

Importance of the C-terminal domain of the human GW182 protein TNRC6C for translational repression

Proteins of the GW182 family play an important role in the execution of microRNA repression in metazoa. They interact directly with Argonaute proteins, components of microRNPs, and also form part of P-bodies, structures implicated in translational repression and mRNA degradation. Recent results demonstrated that Drosophila GW182 has the potential to both repress translation and accelerate mRNA deadenylation and decay. In contrast to a single GW182 protein in Drosophila, the three GW182 paralogs TNRC6A, TNRC6B, and TNRC6C are encoded in mammalian genomes. In this study, we provide evidence that TNRC6C, like TNRC6A and TNRC6B, is important for efficient miRNA repression. We further demonstrate that tethering of each of the human TNRC6 proteins to a reporter mRNA has a dramatic inhibitory effect on protein synthesis. The repression is due to a combination of effects on the mRNA level and mRNA translation. Through deletion and mutagenesis, we identified the C-terminal part of TNRC6C encompassing the RRM RNA-binding motif as a key effector domain mediating protein synthesis repression by TNRC6C.     

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1

MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4–NOT deadenylase complex followed by the removal of the 5′ cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4–NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6–CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.     

A C-terminal silencing domain in GW182 is essential for miRNA function

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182–AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine–tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.