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（逐字附加研究者摘要）：肌强直 营养不良（DM）是成人发病的肌营养不良症的最常见形式。 DM 是 由 (CTG)n 重复序列引起的常染色体显性神经肌肉疾病 DM 蛋白激酶 (DMPK) 基因 3' UTR 的扩展。长期来看 拟议研究的目的是阐明三联体如何重复 基因 3' UTR 的扩展会导致显性遗传性疾病。 目前的证据表明 DM 发病机制与 DMPK 突变等位基因转录物在细胞核内的积累。我们的工作 “隔离”假说认为，DM 是一种 RNA 显性疾病，其中 (CUG)n 扩增形成异常稳定的双链 RNA (dsRNA) 发夹结构。这种不寻常的 RNA 发夹充当高亲和力结合 可能发挥作用的三联体重复扩增 dsRNA 结合蛋白的位点 在核细胞质 RNA 输出中发挥重要作用。大量重复扩展 与严重疾病相关导致这些蛋白质被 DMPK 隔离 突变等位基因转录本和对其他出口的显性负面影响 RNA。该提案的重点是使用以下方法测试该 RNA 优势模型 几种不同的实验方法。首先，假设 (CUG)n 扩增 RNA 对 mRNA 输出具有显着的负面影响，将直接影响 使用 RNA 显微注射到青蛙卵母细胞和哺乳动物成纤维细胞中进行检查 原子核。其次，隔离假说预测扩张结合 蛋白质应与 DMPK 突变体一起积聚在核灶中 成绩单。因此，我们将完成几个特征 优先识别大 (CUG)n 扩展的蛋白质，并确定 这些蛋白质在正常细胞和糖尿病患者细胞中的亚细胞分布。 第三，这些扩展结合蛋白的优选RNA结合位点是 以体外和体内分析为特征，特别强调 识别通常与这些蛋白质相关的RNA。第四，我们将 确定扩展结合蛋白是否参与 mRNA 输出 将单克隆抗体和重组蛋白的使用与 显微注射系统开发的第一个目的。五、RNA的相关性 其他神经肌肉和神经系统疾病的主导地位将是 调查了。这些研究对于阐明 参与 DM 发病机制的分子机制和细胞策略 促进细胞核和细胞核之间遗传信息的交换 细胞质。