RNA Dominance in Human Disease

描述（由申请人提供）：肌强直性营养不良（DM）是由两个不同基因DMPK和ZNF9/CNBP的非编码区CTG和CCTG微卫星扩增引起的。转录后，这些重复扩增折叠成稳定的RNA发夹结构，这是有毒的，因为它们改变了两个可选剪接因子家族的活性，即肌盲样（MBNL）和CUGBP/ et -3样因子（CELF）蛋白。尽管这种rna介导的发病机制解释了成人发病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CTG重复扩增非常大有关。在寻找与CDM发病机制相关的因素时，我们发现Mbnl3的表达仅限于胚胎肌肉发育和出生后肌肉再生。基于这些和其他结果，本研究旨在验证Mbnl3活性对正常肌肉发育和再生至关重要的假设，而Mbnl3活性的丧失导致CDM中肌肉生成延迟，随后导致成人肌肉无力/萎缩。为了验证这一假设，我们产生了Mbnl3条件敲除小鼠，以测试Mbnl3缺失对骨骼肌发育和再生的影响。此外，将在发育和再生肌肉中确定Mbnl3的RNA靶点，随后进行旨在确定Mbnl3缺失如何导致这些靶点的错误调节和特定病理结果的检测。由于我们已经发现Mbnl3编码核蛋白和细胞质蛋白，因此将研究这些同工异构体之间的功能差异，并测试MBNL蛋白在CUG和CCUG扩增rna上形成不同类型复合物的假设。最后，我们将产生Mbnl双重和三重肌肉特异性敲除小鼠来测试我们的模型，即肌强直性营养不良是一种Mbnl功能丧失疾病。这些研究的目标是了解导致CDM和成人发病DM的分子事件，并将这些知识用于开发新的疾病治疗方法。

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.