Johns Hopkins University School of Medicine, Department of Medicine, Division of Rheumatology, Baltimore, Maryland, United States of America.
PLoS One. 2011;6(12):e28861. doi: 10.1371/journal.pone.0028861. Epub 2011 Dec 9.
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.
酵母 Ufd2p 是第一个被鉴定的 E4 多泛素链组装因子。其脊椎动物同源物后来被称为 UFD2a、UBE4B 或 E4B,也被证明具有 E3 泛素连接酶活性。UFD2a 在大脑中的功能在体内已经得到很好的证实,体外研究表明,它的活性对于有丝分裂过程中染色体的适当浓缩和分离是必不可少的。在这里,我们表明 UFD2a 的 2 种替代剪接形式 UFD2a-7 和 -7/7a,在 C2C12 细胞培养物的成肌细胞分化过程中和在小鼠的心脏毒素诱导的骨骼肌再生过程中顺序表达。UFD2a-7 包含一个替代的外显子 7,而 UFD2a-7/7a 是这 2 种异构体中的较大者,包含一个额外的新外显子 7a。对小鼠和斑马鱼的蛋白或 mRNA 表达的分析表明,在心脏和骨骼肌的发育性肌发生过程中,发生了类似的异构体转换模式。在脊椎动物(人类、啮齿动物、斑马鱼)中,UFD2a-7/7a 仅在成熟的横纹肌中表达。这种独特的组织特异性进一步通过位于外显子 7 和 7a 的 3'内含子中的 2 个肌肉特异性剪接调节基序的保守存在得到验证。UFD2a 与 VCP/p97 相互作用,VCP/p97 是一种 AAA 型 ATP 酶,涉及的过程其功能部分通过与一个或多个先前鉴定的 15 个共因子的相互作用来调节。UFD2a-7/7a 在酵母 2 杂交实验中没有与 VCP/p97 相互作用,这可能允许 ATP 酶结合促进其肌肉特异性功能的共因子。我们得出结论,这些 UFD2a 异构体的调节表达很可能赋予了对肌发生很重要的不同功能。