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: 6202115
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202115
• 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基因功能丧失导致脆性X 精神发育迟滞综合症。FMR1编码一种RNA结合蛋白 未知函数。该基因的大多数突变都会导致一种 长度超过1千碱基的多态三核苷酸重复序列。 位于5‘-非翻译部分的这个重复序列的扩展 FMR1基因的缺失与该基因座的甲基化和基因缺失有关 稳定的mRNA水平。生物化学和分子生物学基础 然而，这种失活是未知的，包括特定的dna。 其甲基化导致转录沉默的元件 吉恩。使用核子分析，我们将直接测试是否 转录的启动速度在不同的等位基因中受到损害 重复数和甲基化状态。我们还将测试一种替代方案 假设通过扩展的聚(CGG)转录减少 由于转录受阻而导致FMR1转录产物的输出 延长，就像在其他人类DNA序列中看到的那样，偏离 典型的BDNA结构。在这种情况下，将呈现转录 由伸长系数决定。 FMR1基因产物功能丧失的其他机制包括 描述。FMR1的一个包含点突变的等位基因编码 RNA结合缺陷蛋白。铁磁电阻的三维表面 与RNA接触的蛋白质尚不清楚。在这个项目中，我们将 探讨FMR1致病的基本机制 FMR1转录起始、延长和水平的突变 由FMR蛋白本身结合的RNA。具体目标是： 1)定义作为靶点的顺式作用序列 甲基化介导的启动子功能失活， 2)测量转录速率随 特异性FMR1启动子元件和CGG重复序列的甲基化状态 大小， 3)通过CGG重复序列评估转录作为 三胞胎在体内和体外的扩张程度， 4)对与RNA密切接触的FMRP残基进行定位 纯化的FMRP和修饰的RNA的光交联。这将是 补充FMR基因产物的高分辨率结构研究。 将测试FMR蛋白与RNA接触表面的突变体 在体外和体内发挥作用。