RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) β-Exon Splicing

Kyung-Soon Lee,Yi Cao,Hanna E Witwicka,Susan Tom,Stephen J Tapscott,Edith H Wang

doi:10.1074/jbc.m110.124255

Abstract

Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced β-exon. Expression of the transcriptionally more active (+)β isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 β-exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the CX(7)CX(4-6)CX(3)H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D β-exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.

Highlights

Consistent with the Western blot results, detection of multinucleated myotubes that stained positive for MHC was observed only in control C2C12 cells and in C2C12-MBNL3 cells infected with the (ϩ)␤ isoform of Mef2D (Fig. 3, E, F, K, and L). These findings suggest that Mef2D(ϩ)␤ is essential for muscle differentiation and that the inhibitory effect of MBNL3 on muscle differentiation is due in part to silencing of Mef2D ␤-exon splicing
RNA Binding Activity of MBNL3 Requires the CX7CX4 – 6- we examined the consequences of expressing wild-type CX3H Zinc Finger Motifs—CX4 –15CX4 – 6CX3H zinc finger do- MBNL3 or the four zinc finger mutants on ␤-exon splicing of mains are a feature characteristic of many cellular proteins transcripts derived from the Mef2D minigene (Fig. 6B)
The MBNL proteins are regulators of alternative splicing and have been implicated in the splicing defects associated with the adult onset muscular dystrophy known as myotonic dystrophy

Summary

Introduction

We report here that MBNL3 selectively binds to Mef2D intron 7 sequences and functions as a silencer of ␤-exon splicing during muscle differentiation. In a cell culture model of myotonic dystrophy and DM skeletal muscle tissue, a decrease in Mef2D ␤-exon splicing was accompanied by an increase in MBNL3 expression.

Results

Conclusion