TRANSCRIPTION AND RNA BINDING IN FRAGILE X SYNDROME

脆性 X 综合征中的转录和 RNA 结合

• 批准号: 6108901
• 负责人: Daniel Reines
• 金额: $ 17.25万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6108901
• 关键词: DNA methylation; RNA binding protein; behavioral /social science research tag; behavioral genetics; cell line; crosslink; disease /disorder etiology; fragile X syndromes; gene induction /repression; gene mutation; genetic mapping; genetic promoter element; genetic regulatory element; genetic transcription; genetically modified animals; immunoprecipitation; laboratory rat; nuclear runoff assay; nucleic acid repetitive sequence; sex linked trait; site directed mutagenesis; transcription factor; transfection

Loss of function of the FMR1 gene in humans leads to the Fragile X mental Retardation syndrome. FMR1 encodes an RNA-binding protein of unknown function. Most mutations in the gene result in expansion of a polymorphic trinucleotide repeat to sizes greater than 1 kilobase. Expansion of this repeat, which resides in the 5'-untranslated portion of FMR1, is associated with hypermethylation of the locus and a loss in steady-state levels of mRNA. The biochemical and molecular basis for this inactivation is, however, unknown including the specific DNA elements whose methylation results in transcriptional silencing of the gene. Using nuclear-runon assays, we will test directly whether the rate of initiation of transcription is compromised in alleles of varying repeat number and methylation state. We will also test an alternative hypothesis that transcription through the expanded poly (CGG) reduces the output of FMR1 transcript due to a blockage of transcript elongation, as seen for other human DNA sequences that deviate from the canonical B DNA structure. In such cases, transcription is rendered elongation factor-dependent. Other mechanisms of loss of FMR1 gene product function have been described. One allele of FMR1 containing a point mutation encodes an RNA binding-defective protein. The 3- dimensional surface of FMR protein that contacts RNA is not known. In this project we will investigate the basic mechanisms of disease causation due to FMR1 mutation at the level of FMR1 transcription initiation, elongation, and RNA-binding by the FMR protein itself. The specific aims are: 1) to define the cis-acting sequences that serve as targets for methylation-mediated inactivation of promoter function, 2) to measure transcription rate changes as a function of the methylation state of specific FMR1 promoter elements and the CGG-repeat size, 3) to assess transcription through CGG repeats as a function of the extent of triplet expansion in vivo and in vitro, 4) to map the residues of FMRP that are in close contact with RNA using photo cross-linking of purified FMRP and modified RNA. This will complement high resolution structural studies of the FMR gene product. Mutants in the RNA-contacting surface of the FMR protein will be tested for function in vitro and in vivo.

人类FMR1基因的功能丧失导致脆弱的X 智力障碍综合征。 FMR1编码一种RNA结合蛋白 未知功能。 基因中的大多数突变导致A的扩张 多态三核苷酸重复大于1千座酶。 该重复的扩展，该重复位于5'-非翻译部分 FMR1的of与基因座的高甲基化和损失有关 mRNA的稳态水平。 生化和分子基础 但是，这种灭活是未知的，包括特定的DNA 甲基化导致转录沉默的元素 基因。 使用核跑步测定法，我们将直接测试 在不同的等位基因中，转录的发起率在不同的等位基因中受到损害 重复数和甲基化状态。 我们还将测试替代方案 假设通过扩展的聚（CGG）转录减少 FMR1转录本的输出因转录本的阻塞而导致 伸长，如其他偏离偏离的人DNA序列所见 规范B DNA结构。 在这种情况下，呈现转录 伸长因子依赖性。 FMR1基因产物功能丧失的其他机制已经 描述。 FMR1的一个等位基因包含点突变编码 RNA结合缺陷蛋白。 FMR的3维表面 接触RNA的蛋白质尚不清楚。 在这个项目中，我们将 研究因FMR1引起的疾病因果的基本机制 FMR1转录启动，伸长和 FMR蛋白本身的RNA结合。 具体目的是： 1）定义作为目标的顺式作用序列 甲基化介导的启动子功能失活， 2）测量转录率随着 特定FMR1启动子元素和CGG重复的甲基化状态 尺寸， 3）通过CGG重复评估转录作为函数 体内和体外的三胞胎扩展的程度， 4）绘制使用使用RNA密切接触的FMRP残基 纯化的FMRP和修饰的RNA的照片交联。 这会 补充FMR基因产物的高分辨率结构研究。 将测试FMR蛋白的RNA接触表面中的突变体 用于体外和体内功能。