A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells.

Antony Rosen,Sarah Spinette,Nisha Badders,Irina Kerkis,Andrew L Mammen,Amanda St Germain,James A Mahoney,J Paul Taylor

doi:10.1371/journal.pone.0028861

Abstract

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3′ introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.

Highlights

The way in which cells achieve their differentiation program is intimately tied to changes in their proteome occurring via several mechanisms including, post-translational modification, de novo transcription, ubiquitin dependant proteasomal degradation, and specific regulation of tissue-specific splicing factors resulting in alternatively spliced transcripts
A novel form of UFD2a is expressed in skeletal muscle and cardiac muscle
Western blot analysis revealed that UFD2a expressed in skeletal muscle migrated significantly slower on SDS-PAGE than in other tissues, suggesting a muscle tissue– specific alternate isoform (Fig. 1A)

Summary

Introduction

The way in which cells achieve their differentiation program is intimately tied to changes in their proteome occurring via several mechanisms including, post-translational modification, de novo transcription, ubiquitin dependant proteasomal degradation, and specific regulation of tissue-specific splicing factors resulting in alternatively spliced transcripts. Titin, MEF-2, and MBNL proteins represent specific examples of developmentally regulated splicing events which affect protein function, localization and/ or binding specificity in these tissues [6,7,8,9]. Ubiquitin-dependant degradation facilitates myosin heavy chain (MHC) isoform switching [16] and the is crucial for turnover of myosin binding chaperones that affect overall sarcomere assembly [17,18,19,20]

Methods

Results

Conclusion