MOLECULAR MECHANISMS OF THE MYOTONIC DYSTROPHY MUTATION

肌强直性营养不良（DM）是成人中最常见的遗传性神经肌肉疾病，全球发病率为1 / 8000。发现DM突变是编码丝氨酸-苏氨酸蛋白激酶（DMPK）的基因的3‘非翻译区（3’ utr）中不稳定的CTG三重重复扩增。然而，它引起疾病的机制尚不清楚。我们和其他人已经发现突变的DMPK mRNA被困在DM细胞的细胞核内，并形成独特的、稳定的mRNA焦点。此外，我们已经证明突变DMPK 3'UTR mrna对基因表达有显著的负面影响。此外，我们已经发现DMPK 3'UTR mRNA突变体在成肌细胞中的表达会导致成肌细胞融合和分化的缺陷，这表明该RNA通过反式作用于其他转录物的表达，并导致与疾病相关的细胞表型。本研究旨在通过解决DM是RNA介导的疾病过程范例的假设来理解DM的分子生物学。本实验旨在评估和确定DMPK信使RNA （mRNA）对基因表达的影响。正常和突变DMPK 3'UTR mRNA的影响将首先在细胞水平上进行研究，其次从生化角度进行研究，最后通过建立转基因小鼠模型在体内进行研究。本提案拟验证的假设是：肌强直性营养不良是一种由DMPK mRNA突变介导的RNA代谢失调参与DM病理生理的疾病。本提案的具体目的是：1)在细胞培养模型中研究DM突变的影响；2)鉴定突变DMPK 3' utr mRNA存在后表达改变的基因；3)建立小鼠模型，研究DM突变对体内RNA代谢的影响及其对DM发病机制的贡献。本提案的长期目标是了解糖尿病突变的分子机制，以便深入了解糖尿病的病理生理学，允许开发适当的动物模型，并最终导致更合理的糖尿病治疗干预方法。

Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder in adults with a global incidence of 1 per 8000. The DM mutation was found to be an expansion of an unstable CTG triplet repeat in the 3' untranslated region (3'UTR) of a gene encoding a serine-threonine protein kinase (DMPK). However, the mechanism by which it causes disease is unknown. We and others have found that the mutant DMPK mRNA is trapped within the nucleus of DM cells and forms distinct, stable foci of mRNA. In addition, we have demonstrated that the mutant DMPK 3'UTR mRNAhas significant negative effects on gene expression. Furthermore, we have identified that expression of the mutant DMPK 3'UTR mRNA in myoblasts causes defects in myoblast fusion and differentiation, demonstrating that this RNA work in trans on the expression of other transcripts, and causes a disease relevant cellular phenotype. This study is directed at understanding the molecular biology of DM by addressing the hypothesis that DM is a paradigm for RNA mediated disease processes. The proposed experiments will be aimed at assessing and determining the effect of the DMPK messenger RNA (mRNA) on gene expression. The effects of the normal and mutant DMPK 3'UTR mRNA will be studied initially at the cellular level, secondly from a biochemical persepective and finally in vivo through the creation of a transgenic murine model. The hypothesis to be tested by this proposal is that: Myotonic dystrophy is a disease in which dysregulation of RNA metabolism mediated by the mutant DMPK mRNA contributes to the pathophysiology of DM. The specific aims of this proposal are directed at: 1) studying the effects of the DM mutation in a cell culture model, 2) identifying genes whose expression is altered by the presence of the mutant DMPK 3'UTR mRNA and 3) the establishment of a murine model to study the in vivo effects of the DM mutation on RNA metabolism and their contribution to DM pathogenesis. The long term objectives of this proposal are to understand the molecular mechanisms by which the DM mutation functions in order to provide insight into the pathophysiology of DM, to allow for the development of appropriate animal models, and to eventually lead to a more rational approach to therapeutic intervention in DM.