Molecular Mechanisms of Myotonic Dystrophy

描述(申请人提供)：强直性肌营养不良(DM)是由肌营养不良症肌强直蛋白激酶基因(DMPK)和锌指9基因(ZNF9)非编码区的CUG和CCUG的核苷酸扩张引起的。CUG扩张型患者为I型强直性肌营养不良(DM1)，CCUG扩张型患者为II型强直性肌营养不良(DM2)。DM1和DM2的患者表现出相同的症状，这表明CUG和CCUG的扩张通过相同的机制导致DM。关于这些非编码扩张如何导致DM的假说是通过RNA功能获得机制来实现的；扩展的CUG和CCUG重复RNA隔离了MBNL RNA结合蛋白，并间接增加了另一种RNA结合蛋白(CUG-BP)的蛋白质水平，从而扰乱了MBNL和CUG-BP的正常细胞功能。MBNL和CUG-BP是似乎相互拮抗的前mRNA剪接因子。改变它们的“活性”浓度会导致多个转录本的选择性剪接的错误调节，最终导致患有CUG和CCUG扩张者的DM。这项建议的重点是确定MBNL在疾病状态(DM)以及正常细胞中的作用。为了实现这些目标，我们正在使用生物信息学、生化、生物物理和结构方法的组合。目的1.确定MBNL的细胞靶点，并确定MBNL调节Pre-mRNA剪接的机制。目的2.CUG和CCUG重复序列及其与MBNL的复合体的生化和结构特征。我们的目标是了解糖尿病的分子机制。这一认识将有助于开发治疗方法，帮助许多患有这种最常见的成人发作性肌营养不良症的人(8000人中有1人)。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is caused by nucleotide expansions of CUG and CCUG in the non-coding regions of the dystrophia myotonia protein kinase gene (DMPK) and the Zn finger 9 gene (ZNF9) respectively. Patients with the CUG expansions have type I myotonic dystrophy (DM1) and patients with the CCUG expansions have type II myotonic dystrophy (DM2). Patients with DM1 and DM2 display the same symptoms, suggesting both CUG and CCUG expansions cause DM through the same mechanism. The hypothesis for how these non-coding expansions cause DM is through an RNA gain-of-function mechanism; the expanded CUG and CCUG repeat RNAs sequester the MBNL RNA binding protein and also indirectly increase the protein levels of another RNA binding protein (CUG-BP), which disrupts the normal cellular function of MBNL and CUG-BP. MBNL and CUG-BP are pre-mRNA splicing factors that appear to function antagonistically. Changing their "active" concentration results in the mis-regulation of alternative splicing of multiple transcripts with a final outcome of DM for people with CUG and CCUG expansions. The focus of this proposal is to determine the role of MBNL in the disease state (DM) as well as in normal cells. To accomplish these goals we are using a combination of bioinformatics, biochemical, biophysical and structural methods. Aim 1. Identify cellular targets of MBNL and determine the mechanisms through which MBNL regulates pre- mRNA splicing. Aim 2. Biochemical and structural characterization of CUG and CCUG repeats alone and in complex with MBNL. Our goal is to understand the molecular mechanisms that cause DM. This understanding will help lead to the development of therapies to help the many people (1 in 8000) that have this most common form of adult onset muscular dystrophy.