RNA Dominance in Human Disease

描述（由申请人提供）：强直性肌营养不良（DM）是由两种不同基因DMPK和ZNF 9/CNBP的非编码区中CTG和CCTG微卫星扩增引起的。在转录时，这些重复扩增折叠成稳定的RNA发夹结构，这些发夹结构是有毒的，因为它们改变了两个可变剪接因子家族的活性，即肌盲样（MBNL）和CUGBP/ETR-3样因子（CELF）蛋白。虽然这种RNA介导的发病机制模型解释了成人发病DM的许多方面，但先天性DM（CDM）的分子事件尚不清楚，因为这种形式的疾病仅与DMPK基因中非常大的CTG重复扩增相关。在寻找参与CDM发病机制的因素时，我们发现Mbnl 3表达仅限于胚胎肌肉发育和出生后肌肉再生。基于这些和另外的结果，该提议被设计为测试Mbnl 3活性对于正常肌肉发育和再生是必不可少的假设，并且该活性的丧失导致CDM中的肌生成延迟，随后是成人中的肌肉无力/消瘦。为了测试该假设，已经产生Mbnl 3条件性敲除小鼠以测试Mbnl 3损失对骨骼肌的发育和再生的影响。此外，将在发育和再生肌肉中鉴定Mbnl 3的RNA靶标，然后进行设计用于确定Mbnl 3的损失如何导致这些靶标的误调节和特定病理结果的测定。由于我们已经发现Mbnl 3编码细胞核和细胞质蛋白，因此将研究这些同种型之间的功能差异，并将测试MBNL蛋白在CUG与CCUG扩增RNA上形成不同类型的复合物的假设。最后，我们将产生Mbnl双重和三重肌肉特异性敲除小鼠，以测试我们的模型，强直性肌营养不良是MBNL功能丧失性疾病。这些研究的目的是了解导致CDM和成人发病DM的分子事件，并利用这些知识开发新的疾病治疗方法。 公共卫生相关性：虽然强直性肌营养不良症（DM）是成人中最常见的肌营养不良症形式，但这种疾病也影响新生儿。我们的研究旨在了解导致先天性和成人型DM的疾病相关分子事件。这项研究的重点是了解这种疾病如何影响骨骼肌的发育和再生，这些知识应该使我们能够为这种疾病开发新的治疗方法。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is caused by the expansion of CTG and CCTG microsatellites in the non-coding regions of two different genes, DMPK and ZNF9/CNBP. Upon transcription, these repeat expansions fold into stable RNA hairpin structures that are toxic because they alter the activities of two families of alternative splicing factors, the muscleblind-like (MBNL) and CUGBP/ETR-3-like factor (CELF) proteins. Although this RNA-mediated pathogenesis model accounts for many aspects of adult-onset DM, the molecular events underlying congenital DM (CDM) are less clear since this form of the disease is only associated with very large CTG repeat expansions in the DMPK gene. While searching for factors involved in CDM pathogenesis, we discovered that Mbnl3 expression is restricted to embryonic muscle development and postnatal muscle regeneration. Based on these, and additional, results, this proposal is designed to test the hypothesis that Mbnl3 activity is essential for normal muscle development and regeneration and loss of this activity leads to delayed myogenesis in CDM followed by muscle weakness/wasting in adults. To test this hypothesis, Mbnl3 conditional knockout mice have been generated to test the effects of Mbnl3 loss on the development and regeneration of skeletal muscle. Additionally, RNA targets for Mbnl3 will be identified in both developing and regenerating muscle followed by assays designed to determine how loss of Mbnl3 leads to mis-regulation of these targets and specific pathological outcomes. Since we have discovered that Mbnl3 encodes both nuclear and cytoplasmic proteins, functional differences between these isoforms will be investigated and the hypothesis that MBNL proteins form different types of complexes on CUG versus CCUG expansion RNAs will be tested. Finally, we will generate Mbnl double and triple muscle-specific knockout mice to test our model that myotonic dystrophy is an MBNL loss-of-function disease. The goal of these studies is to understand the molecular events which lead to CDM and adult-onset DM and use this knowledge for the development of novel disease therapies. PUBLIC HEALTH RELEVANCE: While myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults, this disorder also affects newborn children. Our research is directed at understanding the disease-associated molecular events which lead to congenital and adult-onset DM. The study proposed in this grant is specifically focused on understanding how this disease affects the development and regeneration of skeletal muscle and this knowledge should allow us to develop novel therapeutic approaches for this disease.