Molecular Pathogenesis of Myotonic Dystrophy

描述（由申请人提供）：肌强直性营养不良症（DM）是最常见的成人发病肌肉营养不良症，全球8500人中有1人患有此病。它主要是遗传的，影响多个器官系统。致病性突变是在转录但非翻译的基因组区域扩展的三（CTG）和四（CCTG）核苷酸重复。糖尿病的主要致病机制涉及由扩增等位基因的RNA转录物表达引起的毒性RNA功能获得。本研究的目标是确定含有扩增重复序列的RNA导致进行性骨骼肌营养不良和心律失常的分子机制，这是导致死亡率和发病率的主要原因。在之前的资助期内，我们证明了致病机制涉及选择性剪接的错误调节，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，这两个家族都是先前根据CUG RNA结合活性确定的。我们将定义扩展的CUG RNA诱导发病的机制，特别强调这两个蛋白家族的个体成员的作用。首先，虽然很明显，CUG重复RNA是致病性的，但致病性所需的RNA的具体形式尚不清楚。我们将使用一种已建立的测定方法来确定序列、蛋白质结合和RNA的结构特征，这些特征是诱导反式中剪接错误调节所需的。与RNA的直接相互作用与异常RNA加工相关的蛋白质将被识别。其次，将使用新的生化和减法方法识别导致肌病和心律失常的基因。第三，将使用诱导表达扩增CUG RNA的稳定细胞系来表征毒性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系，在特定组织和早期发育中诱导高水平扩增的CUG RNA。这些研究将提供细胞和小鼠模型来定义改变的调控途径和受这种新型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.