The Role of RNA-Binding Proteins in Myogenesis

描述（申请人提供）：骨骼肌的分化和发育是一个复杂的过程，受不同基因表达水平的调控。 两组蛋白质，肌原性转录因子和肌细胞增强因子2（MEF 2），在骨骼肌肌发生中起主要作用。 在骨骼肌成肌过程中调节MEF 2和成肌因子蛋白水平的分子机制尚不清楚。 在这个应用程序中，我们将检查的假设，一个家庭的CUG RNA结合蛋白控制肌肉分化，通过调节MEF 2和MyoD的转录后水平。 该家族的成员之一CUGBP 1在骨骼肌中高度表达，并与神经肌肉疾病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CUGBP 1表达的分析显示，CUGBP 1 mRNA和蛋白水平在分化成肌细胞中显著诱导，表明CUGBP 1在骨骼肌分化中起关键作用。 CUGBP 1在肌发生中的作用也通过对过表达CUGBP 1的小鼠模型的分析得到证实。 CUGBP 1在体内骨骼肌中的非预定表达导致生肌调节因子（p21、肌细胞生成素、MEF 2）的异常表达，其伴随小鼠体重的显著减轻和发育不全。 寻找CUGBP 1依赖性调节肌生成的分子途径，我们发现CUGBP 1与MEF 2A mRNA结合，并且这种结合在细胞培养模型中诱导MEF 2A翻译。 由于CUGBP 1水平在分化的成肌细胞中显著升高，并且由于CUGBP 1诱导MEF 2表达，因此我们提出CUGBP 1通过MEF 2蛋白和生肌因子的翻译控制来调节骨骼肌分化。本申请提出：1）研究在分化过程中诱导CUGBP 1表达的分子机制，并鉴定在分化过程中控制CUGBP 1表达的调节因子（具体目标1）;和2）确定升高的CUGBP 1水平改变骨骼肌中肌源性调节因子表达的分子途径（具体目标2）。非肌肉细胞，小鼠成纤维细胞，将通过MyoD和MEF 2A转化为肌肉细胞。将研究CUGBP 1在肌发生中的MyoD和MEF 2A依赖性途径中的作用。

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.