Functional Analysis of ZNF9 in Myotonic Dystrophy Type 2

描述(申请人提供)：强直性肌营养不良(DM)是成人中最常见的肌肉营养不良形式，大约每8,000人中就有1人受到影响。强直性肌营养不良2型(DM2)是由ZNF9基因第一内含子中四核苷酸重复序列CCTG的扩增引起的。ZNF9的正常生物学功能尚不清楚。已经提出了解释DM2的其他致病机制，包括突变基因的功能丧失和RNA介导的功能获得，其中转录的CCUG重复隔离蛋白的正常功能。我们已经在一个新的蛋白质组筛选中确定了ZNF9，以确定与内部核糖体进入位点(IRES)相关的RNA结合蛋白。IRESS介导不依赖5‘帽复合体的特定mRNAs的翻译起始。我们的功能分析表明，ZNF9是帽非依赖性翻译启动的正调控因子。我们假设ZNF9通常作为一种IRES反式激活因子(ITAF)，用于通过帽非依赖机制翻译的一组人mRNAs。ZNF9中CCTG扩增导致DM2的机制(S)尚不清楚。最近的研究表明，ZNF9的缺失会导致DM2症状。因此，我们计划研究功能丧失模型。这项建议的第一个目标是通过测量DM2患者细胞中的帽非依赖性翻译活动来测试CCTG扩增是否扰乱了ZNF9的正常活动。这一关键问题的答案对于设计治疗干预策略至关重要。我们的初步数据有力地表明，ZNF9在大小写无关的翻译中起到了启动因子的作用。ZNF9调控的人类转录本是未知的。因此，第二个目标是识别与ZNF9相互作用的人类mRNAs。最后，重要的是要了解与ZNF9相互作用的一整套蛋白质，以了解其正常的生物学功能。ZNF9与其他蛋白质的相互作用大多是未知的。因此，第三个目标是利用ZNF9蛋白的亲和纯化和基于高灵敏质谱学的蛋白质组学来鉴定与ZNF9相关的蛋白质。与公共卫生相关。强直性肌营养不良是成人肌营养不良最常见的形式。2型疾病是由ZNF9基因非编码区CCTG重复序列的扩张引起的。本研究旨在探讨ZNF9在2型强直性肌营养不良中的正常和疾病作用。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults, affecting approximately 1 in every 8,000 individuals. Myotonic dystrophy type 2 (DM2) is caused by the expansion of the tetra-nucleotide repeat CCTG in the first intron of the ZNF9 gene. The normal biological function of ZNF9 is unknown. Alternative pathogenic mechanisms have been proposed to explain DM2, including loss of function of the mutated gene and an RNA-mediated gain of function in which the transcribed CCUG repeats sequester proteins from their normal functions. We have identified ZNF9 in a novel proteomic screen to identify RNA binding proteins that associate with an internal ribosome entry site (IRES). IRESs mediate translation initiation of specific mRNAs independent of the 5' cap complex. Our functional assays show that ZNF9 is a positive regulator of cap-independent translation initiation. We hypothesize that ZNF9 normally functions as an IRES trans-activating factor (ITAF) for a population of human mRNAs translated by cap-independent mechanisms. The mechanism(s) by which the CCTG expansion in ZNF9 causes DM2 is poorly understood. Recent studies suggest that loss of ZNF9 results in DM2 symptoms. Hence, we plan to investigate the loss-of-function model. The first goal of this proposal is to test whether the CCTG expansion disrupts the normal activity of ZNF9 by measuring cap-independent translation activity in cells from DM2 patients. The answer to this key question is essential for designing a strategy of therapeutic intervention. Our preliminary data strongly show that ZNF9 functions as an initiation factor for cap-independent translation. Human transcripts regulated by ZNF9 are unknown. Therefore, the second goal is to identify human mRNAs that interact with ZNF9. Finally, it is important to know the complete set of proteins that interact with ZNF9 to understand its normal biological function. The interactions of ZNF9 with other proteins are mostly unknown. Thus, the third goal is to identify proteins that associate with ZNF9 using affinity purification of the ZNF9 protein and highly-sensitive mass spectrometry-based proteomics. PUBLIC HEALTH RELEVANCE. Myotonic dystrophy is the most common form of adult muscular dystrophy. The type 2 form of the disease is caused by expansion of CCTG repeats in the non-coding region of the gene ZNF9. This proposal investigates the normal and disease role of ZNF9 in myotonic dystrophy type 2.