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.     

Divergent GW182 functional domains in the regulation of translational silencing

MicroRNA (miRNA)-mediated gene regulation has become a major focus in many biological processes. GW182 and its long isoform TNGW1 are marker proteins of GW/P bodies and bind to Argonaute proteins of the RNA induced silencing complex. The goal of this study is to further define and distinguish the repression domain(s) in human GW182/TNGW1. Two non-overlapping regions, Δ12 (amino acids 896-1219) containing the Ago hook and Δ5 (amino acids 1670-1962) containing the RRM, both induced comparable silencing in a tethering assay. Mapping data showed that the RRM and its flanking sequences in Δ5, but not the Ago hook in Δ12, were important for silencing. Repression mediated by Δ5 or Δ12 was not differentially affected when known endogenous repressors RCK/p54, GW182/TNGW1, TNRC6B were depleted. Transfected Δ5, but not Δ12, enhanced Ago2-mediated repression in a tethering assay. Transfected Δ12, but not Δ5, released endogenous miRNA reporter silencing without affecting siRNA function. Alanine substitution showed that GW/WG motifs in Δ12 (Δ12a, amino acids 896-1045) were important for silencing activity. Although Δ12 appeared to bind PABPC1 more efficiently than Δ5, neither Δ5 nor Δ12 significantly enhanced reporter mRNA degradation. These different functional characteristics of Δ5 and Δ12 suggest that their roles are distinct, and possibly dynamic, in human GW182-mediated silencing.     

The RRM domain in GW182 proteins contributes to miRNA-mediated gene silencing

Proteins of the GW182 family interact with Argonaute proteins and are required for miRNA-mediated gene silencing. These proteins contain two structural domains, an ubiquitin-associated (UBA) domain and an RNA recognition motif (RRM), embedded in regions predicted to be unstructured. The structure of the RRM of Drosophila melanogaster GW182 reveals that this domain adopts an RRM fold, with an additional C-terminal α-helix. The helix lies on the β-sheet surface, generally used by these domains to bind RNA. This, together with the absence of aromatic residues in the conserved RNP1 and RNP2 motifs, and the lack of general affinity for RNA, suggests that the GW182 RRM does not bind RNA. The domain may rather engage in protein interactions through an unusual hydrophobic cleft exposed on the opposite face of the β-sheet. We further show that the GW182 RRM is dispensable for P-body localization and for interaction of GW182 with Argonaute-1 and miRNAs. Nevertheless, its deletion impairs the silencing activity of GW182 in a miRNA target-specific manner, indicating that this domain contributes to silencing. The conservation of structural and surface residues suggests that the RRM domain adopts a similar fold with a related function in insect and vertebrate GW182 family members.     

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.     

The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression

The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis. Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding, using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role for the GW/WG repeats as effector motifs in miRNA-mediated repression.     

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)‐binding protein (PABP) and the CCR4–NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4–NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4–NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation.     

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.     

Defining a new role of GW182 in maintaining miRNA stability

GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA‐mediated gene silencing. Using lentiviral shRNA‐induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA‐mimic half‐lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA‐mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3′–5′ exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute‐bound miRNA.     

The GW182 protein family in animal cells: New insights into domains required for miRNA-mediated gene silencing

GW182 family proteins interact directly with Argonaute proteins and are required for miRNA-mediated gene silencing in animal cells. The domains of the GW182 proteins have recently been studied to determine their role in silencing. These studies revealed that the middle and C-terminal regions function as an autonomous domain with a repressive function that is independent of both the interaction with Argonaute proteins and of P-body localization. Such findings reinforce the idea that GW182 proteins are key components of miRNA repressor complexes in metazoa.     

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.     

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.     

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.     

Localization of double-stranded small interfering RNA to cytoplasmic processing bodies is Ago2 dependent and results in up-regulation of GW182 and Argonaute-2

Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern, indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA processing and siRNA-mediated silencing through the action of its component proteins.     

siRNA Has Greatly Elevated Mismatch Tolerance at 3′-UTR Sites

It has been noted that target sites located in the coding region or the 3′-untranslated region (3′-UTR) can be silenced to significantly different levels by the same siRNA, but little is known about at what specificity the silencing was achieved. In an exploration of positional effects on siRNA specificity by luciferase reporter system, we surprisingly discovered that siRNA had greatly elevated tolerance towards mismatches in target sites in the 3′-UTR of the mRNA compared with the same target sites cloned in the coding region. Assessment of changes in protein and mRNA levels suggested that the differential mismatch tolerance might have resulted from location-specific translational repression in the 3′-UTR. Ablation of argonaute proteins by AGO-specific siRNAs revealed that the AGO2 had major impact on siRNA silencing activity against sites in both coding region and 3′-UTR, while the silencing of nonnucleolytic AGO proteins (AGO1, AGO3 and AGO4) did not significantly affect silencing of sites in either region. This paper revealed the discovery that the specificity of an siRNA can be affected by the location of its target site.     

Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing

miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A-C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A-C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells.     

mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells

MicroRNA (miRNA) binds to the 3′-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.     

microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation

Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.