RNA DOMINANCE IN HUMAN DISEASE

RNA 在人类疾病中的优势

• 批准号: 6723767
• 负责人: MAURICE SCOTT SWANSON
• 金额: $ 23.14万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723767
• 关键词: RNA binding protein; Xenopus oocyte; autosomal dominant trait; binding sites; disease /disorder etiology; double stranded RNA; intermolecular interaction; messenger RNA; microinjections; molecular pathology; monoclonal antibody; muscular dystrophy; nuclear membrane; nucleic acid repetitive sequence; nucleic acid structure; protein localization; tissue /cell culture

DESCRIPTION (appended verbatim from investigator's abstract): Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. DM is an autosomal dominant neuromuscular disorder that is caused by a (CTG)n repeat expansion in the 3' UTR of the DM protein kinase (DMPK) gene. The long term objective of the proposed research is to elucidate how a triplet repeat expansion in the 3' UTR of a gene leads to a dominantly inherited disease. Current evidence suggests that DM pathogenesis is associated with the accumulation of DMPK mutant allele transcripts within the nucleus. Our working 'sequestration' hypothesis is that DM is an RNA dominant disease in which the (CUG)n expansion forms an exceptionally stable double stranded RNA (dsRNA) hairpin structure. This unusual RNA hairpin acts as a high affinity binding site for triplet repeat expansion dsRNA binding proteins that possibly play important roles in nucleocytoplasmic RNA export. Large repeat expansions associated with severe disease lead to sequestration of these proteins on DMPK mutant allele transcripts and a dominant negative effect on the export of other RNAs. This proposal is focused on testing this RNA dominance model using several different experimental approaches. First, the hypothesis that (CUG)n expansion RNAs have a dominant negative effect on mRNA export will be directly examined using RNA microinjection into frog oocyte and mammalian fibroblast nuclei. Second, the sequestration hypothesis predicts that expansion binding proteins should accumulate in nuclear foci together with DMPK mutant transcripts. Therefore, we will complete the characterization of several proteins that preferentially recognize large (CUG)n expansions, and determine the subcellular distribution of these proteins in normal and DM patient cells. Third, preferred RNA binding sites for these expansion binding proteins will be characterized by in vitro and in vivo analyses with particular emphasis on identifying RNAs that normally associate with these proteins. Fourth, we will determine if expansion binding proteins are involved in mRNA export by combining the use of monoclonal antibodies and recombinant proteins with the microinjection system developed in the first aim. Fifth, the relevance of RNA dominance to other neuromuscular and neurological diseases will be investigated. These studies have important implications for elucidating molecular mechanisms involved in DM pathogenesis and cellular strategies which facilitate the exchange of genetic information between the nucleus and cytoplasm.

描述（研究者摘要逐字化）：myotonic 营养不良（DM）是成人发作肌肉营养不良的最常见形式。 DM是 由A（CTG）n重复引起的常染色体显性神经肌肉疾病 DM蛋白激酶（DMPK）基因的3'UTR中的膨胀。长期 拟议的研究的目的是阐明三胞胎如何重复 基因3'UTR的扩张导致了一种主要遗传的疾病。 当前的证据表明，DM发病机理与 核内DMPK突变等位基因转录本的积累。我们的工作 “隔离”假设是DM是一种RNA主要疾病 （CUG）n膨胀形成了一个异常稳定的双链RNA（DSRNA） 发夹结构。这种不寻常的RNA发夹充当高亲和力结合 三胞胎重复扩展的位点DSRNA结合蛋白可能会播放 在核质RNA输出中的重要作用。大重复扩展 与严重疾病相关的导致这些蛋白质在DMPK上的隔离 突变等位基因转录本和对其他出口的主要负面影响 RNA。该建议的重点是使用 几种不同的实验方法。首先，（cug）n的假设 扩展RNA对mRNA导出具有主要的负面影响 使用RNA显微注射对青蛙卵母细胞和哺乳动物成纤维细胞进行检查 核。其次，隔离假设预测扩展结合 蛋白质应与DMPK突变体一起积聚在核灶中 成绩单。因此，我们将完成几个的表征 优先识别大型（CUG）N扩展的蛋白质，并确定 这些蛋白质在正常和DM患者细胞中的亚细胞分布。 第三，这些扩展结合蛋白的首选RNA结合位点将是 以体外和体内分析为特征，特别强调 识别通常与这些蛋白相关的RNA。第四，我们会的 确定通过 将单克隆抗体和重组蛋白的使用结合使用 在第一个目标中发展了微注射系统。第五，RNA的相关性 对其他神经肌肉和神经系统疾病的主导地位将是 调查。这些研究对阐明具有重要意义 参与DM发病机理和细胞策略的分子机制，这些机制 促进核之间遗传信息的交换 细胞质。