Phase Transitions in the Assembly and Function of Human miRISC.

miRISC is a multi-protein assembly that uses microRNAs (miRNAs) to identify mRNAs targeted for repression. Dozens of miRISC-associated proteins have been identified, and interactions between many factors have been examined in detail. However, the physical nature of the complex remains unknown. Here, we show that two core protein components of human miRISC, Argonaute2 (Ago2) and TNRC6B, condense into phase-separated droplets in vitro and in live cells. Phase separation is promoted by multivalent interactions between the glycine/tryptophan (GW)-rich domain of TNRC6B and three evenly spaced tryptophan-binding pockets in the Ago2 PIWI domain. miRISC droplets formed in vitro recruit deadenylation factors and sequester target RNAs from the bulk solution. The condensation of miRISC is accompanied by accelerated deadenylation of target RNAs bound to Ago2. The combined results may explain how miRISC silences mRNAs of varying size and structure and provide experimental evidence that protein-mediated phase separation can facilitate an RNA processing reaction.

Convergence of multiple RNA-silencing pathways on GW182/TNRC6.

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.

miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms.

MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.

RNAi in cell nuclei: potential for a new layer of biological regulation and a new strategy for therapeutic discovery.

RNA interference is almost always associated with post-transcriptional silencing in the cytoplasm. MicroRNAs (miRNAs) and critical RNAi protein factors like argonaute (AGO) and trinucleotide repeat binding containing 6 protein (TNRC6), however, are also found in cell nuclei, suggesting that nuclear miRNAs may be targets for gene regulation. Designed small duplex RNAs (dsRNAs) can modulate nuclear processes such as transcription and splicing, suggesting that they can also provide leads for therapeutic discovery. The goal of this Perspective is to provide the background on nuclear RNAi necessary to guide discussions on whether nuclear RNAi can play a role in therapeutic development programs.

Multivalent Recruitment of Human Argonaute by GW182.

In miRNA-mediated gene silencing, the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of the targeted mRNA. GW182 can interact with hAgo via three of the GW/WG repeats in its Argonaute-binding domain: motif-1, motif-2, and the hook motif. The structure of hAgo1 in complex with the hook motif of hGW182 reveals a "gate"-like interaction that is critical for GW182 docking into one of hAgo1's tryptophan-binding pockets. We show that hAgo1 and hAgo2 have a single GW182-binding site and that miRNA binding increases hAgo's affinity to GW182. With target binding occurring rapidly, this ensures that only mature RISC would be recruited for silencing. Finally, we show that hGW182 can recruit up to three copies of hAgo via its three GW motifs. This may explain the observed cooperativity in miRNA-mediated gene silencing.

Distinct temporal expression of the GW182 paralog TNRC6A in neurons regulates dendritic arborization.

Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

N-terminal Ago-binding domain of GW182 contains a tryptophan-rich region that confer binding to the CCR4-NOT complex.

GW182 family proteins are a key component of microRNA-protein complex eliciting translational repression and/or degradation of microRNA-targets. The microRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4-NOT complex which accelerates deadenylation and inhibits translation. The carboxyl-terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4-NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino-terminal Argonaute-binding domain of human GW182/TNRC6A can associate with the CCR4-NOT complex. This region resides between the two Argonaute-binding sites and contains reiterated GW/WG-motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4-NOT complex. Furthermore, co-expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4-NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4-NOT complex.

Impact of scaffolding protein TNRC6 paralogs on gene expression and splicing.

TNRC6 is a scaffolding protein that bridges interactions between small RNAs, argonaute (AGO) protein, and effector proteins to control gene expression. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C These paralogs have ∼40% amino acid sequence identity and the extent of their unique or redundant functions is unclear. Here, we use knockout cell lines, enhanced crosslinking immunoprecipitation (eCLIP), and high-throughput RNA sequencing (RNA-seq) to explore the roles of TNRC6 paralogs in RNA-mediated control of gene expression. We find that the paralogs are largely functionally redundant and changes in levels of gene expression are well-correlated with those observed in AGO knockout cell lines. Splicing changes observed in AGO knockout cell lines are also observed in TNRC6 knockout cells. These data further define the roles of the TNRC6 isoforms as part of the RNA interference (RNAi) machinery.

Cellular RNA Hubs: Friends and Foes of Plant Viruses.

RNA granules are dynamic cellular foci that are widely spread in eukaryotic cells and play essential roles in cell growth and development, and immune and stress responses. Different types of granules can be distinguished, each with a specific function and playing a role in, for example, RNA transcription, modification, processing, decay, translation, and arrest. By means of communication and exchange of (shared) components, they form a large regulatory network in cells. Viruses have been reported to interact with one or more of these either cytoplasmic or nuclear granules, and act either proviral, to enable and support viral infection and facilitate viral movement, or antiviral, protecting or clearing hosts from viral infection. This review describes an overview and recent progress on cytoplasmic and nuclear RNA granules and their interplay with virus infection, first in animal systems and as a prelude to the status and current developments on plant viruses, which have been less well studied on this thus far.

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.

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. METHODS: We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3'UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples. RESULTS: For each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3'UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region. CONCLUSIONS: Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3'UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.

MicroRNP-mediated translational activation of nonadenylated mRNAs in a mammalian cell-free system.

MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4-NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let-7-microRNP-mediated translational activation of nonadenylated target mRNAs in a mammalian cell-free system, which contains over-expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4-1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)-binding protein (PABP) interacting motif (PAM2) from the TNRC6B-SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP-mediated mRNA regulation.

The AGO proteins: an overview.

Small RNAs govern almost every biological process in eukaryotes associating with the Argonaute (AGO) proteins to form the RNA-induced silencing complex (mRISC). AGO proteins constitute the core of RISCs with different members having variety of protein-binding partners and biochemical properties. This review focuses on the AGO subfamily of the AGOs that are ubiquitously expressed and are associated with small RNAs. The structure, function and role of the AGO proteins in the cell is discussed in detail.

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA.

MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3' untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase-RAC-alpha serine/threonine-protein kinase-mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.

Early origin and adaptive evolution of the GW182 protein family, the key component of RNA silencing in animals.

The GW182 proteins are a key component of the miRNA-dependent post-transcriptional silencing pathway in animals. They function as scaffold proteins to mediate the interaction of Argonaute (AGO)-containing complexes with cytoplasmic poly(A)-binding proteins (PABP) and PAN2-PAN3 and CCR4-NOT deadenylases. The AGO-GW182 complexes mediate silencing of the target mRNA through induction of translational repression and/or mRNA degradation. Although the GW182 proteins are a subject of extensive experimental research in the recent years, very little is known about their origin and evolution. Here, based on complex functional annotation and phylogenetic analyses, we reveal 448 members of the GW182 protein family from the earliest animals to humans. Our results indicate that a single-copy GW182/TNRC6C progenitor gene arose with the emergence of multicellularity and it multiplied in the last common ancestor of vertebrates in 2 rounds of whole genome duplication (WGD) resulting in 3 genes. Before the divergence of vertebrates, both the AGO- and CCR4-NOT-binding regions of GW182s showed significant acceleration in the accumulation of amino acid changes, suggesting functional adaptation toward higher specificity to the molecules of the silencing complex. We conclude that the silencing ability of the GW182 proteins improves with higher position in the taxonomic classification and increasing complexity of the organism. The first reconstruction of the molecular journey of GW182 proteins from the ancestral metazoan protein to the current mammalian configuration provides new insight into development of the miRNA-dependent post-transcriptional silencing pathway in animals.

Identification of Phosphorylated Amino Acids in Human TNRC6A C-Terminal Region and Their Effects on the Interaction with the CCR4-NOT Complex.

Human GW182 family proteins have Argonaute (AGO)-binding domains in their N-terminal regions and silencing domains, which interact with RNA silencing-related proteins, in their C-terminal regions. Thus, they function as scaffold proteins between the AGO protein and RNA silencing-related proteins, such as carbon catabolite repressor4-negative on TATA (CCR4-NOT) or poly(A)-binding protein (PABP). Our mass spectrometry analysis and the phosphorylation data registered in PhosphoSitePlus, a post-translational modification database, suggested that the C-terminal region of a human GW182 family protein, TNRC6A, has at least four possible phosphorylation sites, which are located near the region interacting with the CCR4-NOT complex. Among them, two serine residues at amino acid positions 1332 and 1346 (S1332 and S1346) were certainly phosphorylated in human HeLa cells, but other two serine residues (S1616 and S1691) were not phosphorylated. Furthermore, it was revealed that the phosphorylation patterns of TNRC6A affect the interaction with the CCR4-NOT complex. When S1332 and S1346 were dephosphorylated, the interactions of TNRC6A with the CCR4-NOT complex were enhanced, and when S1616 and S1691 were phosphorylated, such interaction was suppressed. Thus, phosphorylation of TNRC6A was considered to regulate the interaction with RNA silencing-related factors that may affect RNA silencing activity.

GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of MicroRNAs in Mammalian Cancer Cells.

MicroRNAs (miRNAs) are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turnover in cancer cells, we explored the effect of target mRNAs on the abundance of the miRNAs that repress them. We have noted an accelerated extracellular vesicle (EV)-mediated export of miRNAs in presence of their target mRNAs in mammalian cells, and this target-driven miRNA-export process is retarded by Ago2-interacting protein GW182B. The GW182 group of proteins are localized to GW182 bodies or RNA processing bodies in mammalian cells, and GW182B-dependent retardation of miRNA export depends on GW body integrity and is independent of the HuR protein-mediated auxiliary pathway of miRNA export. Our data thus support the existence of a HuR-independent pathway of miRNA export in human cells that can be targeted in MDA-MB-231 cancer cells, to increase the level of cellular let-7a, a known negative regulator of cancer growth.

Argonaute proteins: Structural features, functions and emerging roles.

Argonaute proteins are highly conserved in almost all organisms. They not only involve in the biogenesis of small regulatory RNAs, but also regulate gene expression and defend against foreign pathogen invasion via small RNA-mediated gene silencing pathways. As a key player in these pathways, the abnormal expression and/or mis-modifications of Argonaute proteins lead to the disorder of small RNA biogenesis and functions, thus influencing multiply biological processes and disease development, especially cancer. In this review, we focus on the post-translational modifications and novel functions of Argonaute proteins in alternative splicing, host defense and genome editing.

Role of GW182 protein in the cell.

GW182 proteins interact directly with the argonaute proteins and constitute key components of miRNA repressor complexes (miRISC) in metazoans. As argonautes are insufficient for silencing they recruit the GW182 s that act as scaffold proteins inducing downstream translational repression, target mRNA deadenylation and exonucleolytic mRNA degradation. Besides their role as part of repressor complexes inside the cell, they function in wide variety of cellular processes as highlighted in this review. The present review summarises and discusses in detail our current knowledge of the GW182 s and their role inside the cell.