Molecular Pathogenesis of Myotonic Dystrophy

描述(由申请人提供)：强直性肌营养不良(DM)是最常见的成人型肌营养不良症，全世界每8500人中就有一人患有此病。它主要是遗传的，影响多个器官系统。致病突变是在转录的但非翻译的基因组区域中扩大的三(CTG)和四(CCTG)核苷酸重复。DM的一个主要致病机制涉及到由扩增的等位基因的RNA转录本的表达引起的有毒的RNA功能获得。这项建议的目标是确定包含扩展重复序列的RNA导致进行性骨骼肌营养不良和心律失常的分子机制，这是导致死亡和发病率的主要原因。在之前的资助期间，我们证明了致病机制涉及选择性剪接的错误调控，并确定了导致特定症状的靶基因。我们还发现，这些靶点受到两个RNA结合蛋白家族的拮抗调节，这两个家族都是基于CUG RNA结合活性而鉴定的。我们将明确扩展的CUG RNA诱导发病的机制，特别强调这两个蛋白家族的单个成员的作用。首先，虽然很明显CUG重复RNA是致病的，但致病所需的RNA的特定形式尚不清楚。我们将使用建立的分析方法来定义诱导反式剪接误调控所需的RNA的序列、蛋白质结合和结构特征。与RNA的直接相互作用与异常的RNA加工相关的蛋白质将被识别。其次，将使用新的生化和消减方法来识别其错误调节导致肌病和心律失常的基因。第三，稳定表达扩增的CUG RNA的细胞系将被用来表征有毒RNA的分布和代谢，以及对与细胞毒性相关的RNA结合蛋白表达的影响。第四，我们将使用Cre/loxP策略开发多用途转基因小鼠品系，以在特定组织和早期发育中诱导高水平的扩展CUG RNA。这些研究将提供细胞和小鼠模型，以确定改变的调控途径和受这种新疾病机制影响的基因。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy affecting 1 in 8500 people worldwide. It is dominantly inherited and affects multiple organ systems. Causative mutations are expanded tri-(CTG) and tetra-(CCTG) nucleotide repeats in transcribed but non-translated genomic regions. A major pathogenic mechanism of DM involves a toxic RNA gain-of-function caused by expression of RNA transcripts from the expanded alleles. The goal in this proposal is to determine the molecular mechanism by which RNA containing the expanded repeats causes progressive skeletal muscle dystrophy and cardiac arrhythmias, the predominant causes of mortality and morbidity. In the previous funding period we demonstrated that the pathogenic mechanism involves misregulation of alternative splicing and we identified target genes responsible for specific symptoms. We also found, that these targets are regulated antagonistically by two families of RNA binding proteins both identified previously based on CUG RNA binding activity. We will define the mechanism by which expanded CUG RNA induces pathogenesis with specific emphasis on the roles of individual members of these two protein families. First, while it is clear that CUG repeat RNA is pathogenic, the specific form of the RNA required for pathogenicity is unknown. We will use an established assay to define the sequence, protein binding, and structural features of RNA required for induction of splice-misregulation in trans. Proteins whose direct interactions with the RNA correlate with aberrant RNA processing will be identified. Second, genes whose mis-regulation contributes to myopathy and arrhythmias will be identified using novel biochemical and subtractive approaches. Third, stable cell lines inducibly expressing expanded CUG RNA will be used to characterize the distribution and metabolism of toxic RNA and the consequences on the expression of the RNA binding proteins that are relevant to cell toxicity. Fourth, we will develop versatile lines of transgenic mice using a Cre/LoxP strategy to induce high levels of expanded CUG RNA in specific tissues and in early development. These studies will provide cellular and mouse models to define altered regulatory pathways and the genes affected by this novel disease mechanism.