MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells.

B Cardinali,D Lazarevic,J M Garcia-Manteiga,C Provenzano,F Martelli,D Cittaro,G Falcone,M Cappella

doi:10.1038/cddis.2016.10

Abstract

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein.

Highlights

A number of miRNAs have been implicated in myogenesis and muscle disease, some expressed in muscle cells, others ubiquitously expressed.[2,3] Two closely related miRNAs, miR-221 and miR-222, were previously shown to be downmodulated during differentiation and to induce a delay in progression of differentiation and alterations of myotube morphology and contractile structures when overexpressed.[4]
In order to identify among the predicted miR-222 targets those involved in skeletal myogenesis, we combined in silico target prediction with RNA-induced silencing complex (RISC)-immunoprecipitated Ago2 protein (IP) followed by next-generation sequencing of co-precipitated RNAs17–19 using primary mouse satellite cells (MSC) ectopically expressing miR-222, as myogenic cell model
We have developed a target immunopurification method based on the immunoprecipitation of endogenous RISC complexes enriched for miR-222 and its target mRNAs, using antibodies specific for Ago[2], a core component of the RISC

Summary

Introduction

A number of miRNAs have been implicated in myogenesis and muscle disease, some expressed in muscle cells, others ubiquitously expressed.[2,3] Two closely related miRNAs, miR-221 and miR-222, were previously shown to be downmodulated during differentiation and to induce a delay in progression of differentiation and alterations of myotube morphology and contractile structures when overexpressed.[4]. In order to identify among the predicted miR-222 targets those involved in skeletal myogenesis, we combined in silico target prediction with RISC-IP followed by next-generation sequencing of co-precipitated RNAs17–19 using primary mouse satellite cells (MSC) ectopically expressing miR-222, as myogenic cell model This approach allowed us to discover and functionally validate a number of miR-222 target transcripts. We show here that Rbm[24], a muscle-specific RNA-binding protein having a major role in regulation of muscle development and differentiation[20,21,22,23] is a direct target of miR-222 and its inhibition by miR-222 impairs muscle-specific alternative splicing

Methods

Results

Conclusion