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The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing.

ABSTRACT: 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.

SUBMITTER: Lian SL

PROVIDER: S-EPMC2673069 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing.

Lian Shang L SL Li Songqing S Abadal Grant X GX Pauley Brad A BA Fritzler Marvin J MJ Chan Edward K L EK

RNA (New York, N.Y.) 20090326 5

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. ...[more]

PMID: 19324964

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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.

Dataset Information

The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing.

Publications

The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing.

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