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

Giovanni Perconti,Michele Tumminello,Flavia Contino,Salvatore Feo,Agata Giallongo,Patrizia Rubino,Claudia Coronnello,Giorgio Bertolazzi,Serena Bivona

doi:10.1186/s12859-019-2683-y

Abstract

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.

Highlights

MicroRNAs are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell
While miRNAs guide AGOs to target mRNAs, a direct interaction between AGO and GW182 proteins is required for the assembly of ribonucleoprotein complexes, named RNA-induced silencing complex (RISC), and the recruitment of additional factors involved in gene silencing, which is achieved through the degradation of target mRNAs or translational repression [3, 4]
AGO2 and GW182 proteins complexes handle different mRNA content To gain new insight into the regulatory networks of gene expression involving functionally diverse RISCs in the cell cytoplasm, we used RNA-binding protein immunoprecipitation (RIP)-Chip to identify mRNAs and miRNAs selectively bound to these complexes in the MCF-7 cell line, which is widely used and representative of luminal breast cancer

Summary

Introduction

MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Argonaute (AGO) proteins and the GW182 protein family ( known as TNRC6 proteins) are involved in the cellular process which leads to gene silencing mediated by miRNAs, small endogenous non-coding RNAs that act as post-transcriptional regulators by base pairing to target mRNAs [1, 2]. AGOs, together with GW182/TNRC6A and its mammalian paralogs, TNRC6B and TNRC6C, have a role in executing miRNA-mediated repression, either by silencing or decay, but the proteins contribute to other functions in the nucleus, such as transcription and splicing control [6, 7]. It has been shown that these bodies contain proteins involved in diverse post-transcriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay, translational repression, RNA-mediated gene silencing, and may function as a cytoplasmic domain for RNA storage

Objectives

Methods

Results

Discussion

Conclusion