MyoD Is Indispensable for Muscle-specific Alternative Splicing in Mouse Mitochondrial ATP Synthase γ-Subunit Pre-mRNA

Masaru Ichida,Hitoshi Endo,Uichi Ikeda,Chie Matsuda,Eriko Ueno,Kazuyuki Shimada,Yasuo Kagawa

doi:10.1074/jbc.273.14.8492

Abstract

Muscle-specific alternative RNA splicing is an essential step during myogenesis. In this paper, we report that a muscle-specific transcription factor, MyoD, plays a central role in the induction of muscle-specific alternative splicing during myogenesis. Recently, we reported that muscle and nonmuscle isoforms of the mitochondrial ATP synthase gamma-subunit (F1gamma) were generated by alternative splicing and that acidic stimulation promoted this muscle-specific alternative splicing (Endo, H., Matsuda, C., and Kagawa, Y. (1994) J. Biol. Chem. 269, 12488-12493). In this report, mouse myoblasts are shown to express the muscle-specific isoform of F1gamma after induction with low-serum medium (differentiation medium) or acidic medium, although myotube formation was not detected after acidic induction. RNA blot analysis revealed that the expression levels of both MEF2 and myogenin were increased by low-serum induction, but not by acidic induction. High expression of MyoD mRNA was observed after both types of induction. Overexpression of exogenous MyoD in fibroblasts showed that MyoD was necessary for muscle-specific alternative splicing in both types of induction. Exogenous Id, a negative regulator of MyoD, blocked muscle-specific alternative splicing of F1gamma pre-mRNA by both types of induction. In addition, MyoD induced several muscle-specific alternative splicings, including structural protein pre-mRNAs such as beta-tropomyosin and neural-cell adhesion molecule and transcriptional protein pre-mRNAs such as MEF2A and MEF2D. Our analysis of the two induction systems shows a common MyoD-dependent mechanism of muscle-specific alternative splicing in several genes, independent of MEF2 and myogenin.

Highlights

Alternative pre-mRNA splicing is a fundamental process in eukaryotes and is regulated by cell-specific, tissue-specific, and developmental stage-specific pathways to generate mRNAs that differ in protein-coding potential, stability, and translation efficacy [1, 2]
We show the new muscle-specific alternative splicing induction system using mouse myoblasts, in which acidic stimulation induces muscle-specific splicing without expression of Myocyte-specific enhancer factor 2 (MEF2) and myogenin and without myotube formation
We showed that acidic stimulation induces muscle-specific alternative splicing in F1␥ pre-mRNA and in other muscle-specific pre-mRNAs in mouse myoblasts (C2C12 cells) and does not induce gene expression of MEF2 and myogenin and myotube formation

Summary

Introduction

Alternative pre-mRNA splicing is a fundamental process in eukaryotes and is regulated by cell-specific, tissue-specific, and developmental stage-specific pathways to generate mRNAs that differ in protein-coding potential, stability, and translation efficacy [1, 2]. Understanding the regulatory cascade for tissue-specific alternative splicing during terminal differentiation would elucidate a direct trigger for the alternative splicing mechanism. Myogenic differentiation involves both muscle-specific RNA processing and transcriptional activation of muscle-specific genes, e.g. the basic helix-loop-helix (bHLH) proteins of the MyoD family including MyoD, Myf, MRF4, and myogenin, which promote skeletal muscle-specific gene expression (6 –17) and are built up to a positive autoregulatory loop (6 –12). A recent study shows that the MEF2 and MyoD families act within a regulatory network that establishes differentiated phenotypes of skeletal muscle and that MEF2 factors act as coregulators to potentiate the myogenic activities of myogenic bHLH proteins [26]

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