Similar Datasets
Project description:BackgroundMicroRNAs (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.MethodsWe 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.ResultsFor 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.ConclusionsDue 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.
| S-EPMC6471694 | biostudies-literature
Project description:MicroRNAs 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 complex functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibody and compiled a dataset 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 predicted 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 differentially expressed genes, independently for AGO2 and GW182 related samples. For each of the two proteins, we trained and tested several support vector machine algorithms able to predict the differentially expressed genes that were experimentally detected. The most efficient algorithm for predicting the over-expressed 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 predicting over-expressed genes in the GW182 immunoprecipitated sample was the length of the coding region. Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC complex recruits genes based on miRNA binding sites in the 3’UTR and coding region, but only the longer mRNAs remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.
2018-09-05 | GSE109665 | GEO
Project description:MicroRNAs 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 complex functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibody and compiled a dataset 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 predicted 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 differentially expressed genes, independently for AGO2 and GW182 related samples. For each of the two proteins, we trained and tested several support vector machine algorithms able to predict the differentially expressed genes that were experimentally detected. The most efficient algorithm for predicting the over-expressed 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 predicting over-expressed genes in the GW182 immunoprecipitated sample was the length of the coding region. Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC complex recruits genes based on miRNA binding sites in the 3’UTR and coding region, but only the longer mRNAs remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.
2018-09-05 | GSE109666 | GEO
Project description:RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets
| PRJNA431613 | ENA
Project description:RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets (microRNA)
| PRJNA431617 | ENA
Project description:RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets (mRNA)
| PRJNA431618 | ENA
Project description:MicroRNA (miRNA)-mediated silencing is a post-transcriptional mechanism that regulates translation of mRNAs primarily via their 3'-UTR. Ago2 binds miRNA directly and is the core component of miRNA-induced silencing complex. GW182 is another important factor in miRNA-mediated silencing, and its interaction with Ago2 is evolutionarily conserved. However, the GW182-Ago2 interaction in humans has not been characterized thoroughly, and the role of GW182 in the mammalian miRNA pathway remains unclear. In the current study, we generated a set of GST-, green fluorescence protein (GFP)-, or 3xFlag-tagged deletion constructs of GW182 and Ago2 to further analyze GW182-Ago2 interactions. The C-terminal half of Ago2 interacted with four nonoverlapping GW-rich regions of GW182, and this interaction recruited Ago2 to GWB. Furthermore, the interaction with GW182 was observed in all four human Ago proteins. Most interestingly, tethering the C-terminal half of Ago2 to the 3'-UTR of reporter mRNA recapitulated translational repression comparable to that of tethered Ago2, and this repression was greatly impaired upon GW182 knockdown. In comparison, the N-terminal half of Ago2 did not bind GW182 and did not retain the repression function of Ago2. Our data strongly support a model in which Ago2 recruits GW182 to the 3'-UTR of mRNA to mediate silencing, and suggest that GW182 may contribute to enhancement in translational repression by interacting with multiple Ago proteins from multiple miRNA target sites in the same or adjacent 3'UTR.
| S-EPMC2673069 | biostudies-literature
Project description: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.
| S-EPMC3616289 | biostudies-literature
Project description: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.
| S-EPMC2613116 | biostudies-literature
Project description:MicroRNAs (miRNAs) repress target genes through a poorly defined antisense mechanism. Cell-free and cell-based assays have supported the idea that miRNAs repress their target mRNAs by blocking initiation of translation, whereas studies in animal models argued against this possibility. We examined endogenous targets of the let-7 miRNA, an important regulator of stem cell fates. We report that let-7 represses translation initiation in Caenorhabditis elegans, demonstrating this mode of action for the first time in an organism. Unexpectedly, although the lin-4 miRNA was previously reported to repress its targets at a step downstream of translation initiation, we also observe repression of translation initiation for this miRNA. This repressive mechanism, which frequently but not always coincides with transcript degradation, requires the GW182 proteins AIN-1 and AIN-2, and acts on several mRNAs targeted by different miRNAs. Our analysis of an expanded set of endogenous miRNA targets therefore indicates widespread repression of translation initiation under physiological conditions and establishes C. elegans as a genetic system for dissection of the underlying mechanisms.
| S-EPMC2637332 | biostudies-literature