The Role of RNA-Binding Proteins in Myogenesis

描述（由申请人提供）：骨骼肌的分化和发育是一个复杂的过程，受不同水平基因表达的调控。两组蛋白，肌生成转录因子和肌细胞增强因子2 (MEF2)，在骨骼肌肌生成中起主要作用。调控骨骼肌肌生成过程中MEF2蛋白水平和肌生成因子的分子机制尚不清楚。在这个应用中，我们将检验CUG rna结合蛋白家族通过调节MEF2和MyoD在转录后水平上控制肌肉分化的假设。该家族的成员之一CUGBP1在骨骼肌中高表达，并与肌强直性营养不良患者骨骼肌分化的延迟有关。对分化过程中CUGBP1表达的分析显示，在成肌细胞分化过程中，CUGBP1 mRNA和蛋白水平显著诱导，提示CUGBP1在骨骼肌分化过程中发挥关键作用。CUGBP1在肌肉发生中的作用也通过过表达CUGBP1的小鼠模型分析得到证实。体内骨骼肌中CUGBP1的非预定表达会导致肌生成调节因子（p21, myogenin, MEF2）的异常表达，并伴有小鼠体重的显著减轻和发育不足。在寻找cugbpl依赖性调节肌发生的分子途径时，我们发现CUGBP1与MEF2A mRNA结合，并在细胞培养模型中诱导MEF2A翻译。由于CUGBP1水平在分化成肌细胞中显著升高，并且由于CUGBP1诱导MEF2表达，我们提出CUGBP1通过翻译控制MEF2蛋白和成肌因子来调节骨骼肌分化。本申请拟：1)研究诱导分化过程中CUGBP1表达的分子机制，确定分化过程中控制CUGBP1表达的调控因子（Specific Aim 1）；2)确定CUGBP1水平升高改变骨骼肌中肌生成调节因子表达的分子途径（Specific Aim 2）。非肌肉细胞，小鼠成纤维细胞，将通过MyoD和MEF2A转化为肌肉细胞。CUGBP1在MyoD和MEF2A依赖通路中肌发生的作用将被研究。

DESCRIPTION (provided by applicant): Differentiation and development of skeletal muscle are complex processes that are regulated at different levels of gene expression. Two groups of proteins, myogenic transcription factors and myocyte enhancer factors 2 (MEF2), play a major role in skeletal muscle myogenesis. Molecular mechanisms, which regulate protein levels of MEF2 and myogenic factors during skeletal muscle myogenesis, are not well understood. In this application, we will examine the hypothesis that a family of CUG RNA-binding proteins controls muscle differentiation through the regulation of MEF2 and MyoD on post-transcription levels. One of the members of this family, CUGBP1, is highly expressed in skeletal muscle and is implicated in the delay of skeletal muscle differentiation in patients with a neuro-muscular disease, Myotonic Dystrophy. Analysis of CUGBP1 expression during differentiation course revealed a dramatic induction of CUGBP1 mRNA and protein levels in differentiating myoblasts, suggesting a key role of CUGBP1 in differentiation of skeletal muscle. The role of CUGBP1 in myogenesis is also confirmed by the analysis of mouse model overexpressing CUGBP1. The unscheduled expression of CUGBP1 in skeletal muscle in vivo leads to an abnormal expression of myogenic regulators (p21, myogenin, MEF2) that is accompanied by significant loss of mouse weight and by underdevelopment. Searching for molecular pathways of CUGBPl-dependent regulation of myogenesis, we found that CUGBP1 binds to MEF2A mRNA and that this binding induces MEF2A translation in cell culture models. Since CUGBP1 levels are dramatically elevated in differentiated myoblasts and because CUGBP1 induces MEF2 expression, we propose that CUGBP 1 regulates skeletal muscle differentiation through translational control of MEF2 proteins and myogenic factors. This application proposes: 1) to study molecular mechanisms responsible for induction of CUGBP1 expression during differentiation and identify regulatory factors controlling CUGBP1 expression during differentiation (Specific Aim 1); and 2) to determine molecular pathway by which elevated levels of CUGBP1 alter expression of myogenic regulators in skeletal muscle (Specific Aim 2). Non-muscle cells, mouse fibroblasts, will be converted into muscle cells by MyoD and MEF2A. The role of CUGBP1 in the MyoD and MEF2A dependent pathways in myogenesis will be investigated.