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 基因功能丧失会导致脆性 X 细胞 精神发育迟滞综合症。 FMR1 编码 RNA 结合蛋白 未知功能。 大多数基因突变都会导致 多态性三核苷酸重复长度大于 1 KB。 该重复序列的扩展，位于 5'-非翻译部分 FMR1 的，与位点的高甲基化和缺失相关 mRNA 的稳态水平。 生化和分子基础 然而，这种失活是未知的，包括特定的 DNA 甲基化导致转录沉默的元件 基因。 使用核运行分析，我们将直接测试是否 不同等位基因的转录起始率受到影响 重复次数和甲基化状态。 我们还将测试替代方案 假设通过扩展的多聚 (CGG) 转录会减少 由于转录本阻塞而导致 FMR1 转录本的输出 延伸，正如其他人类 DNA 序列偏离 典型的 B DNA 结构。 在这种情况下，转录被渲染 伸长因子依赖性。 FMR1基因产物功能丧失的其他机制已被证实 描述的。 FMR1 的一个等位基因含有点突变，编码 RNA 结合缺陷蛋白。 FMR的3维表面 与 RNA 接触的蛋白质尚不清楚。 在这个项目中我们将 研究 FMR1 致病的基本机制 FMR1 转录起始、延伸和水平的突变 FMR 蛋白本身与 RNA 结合。 具体目标是： 1) 定义作为靶标的顺式作用序列 甲基化介导的启动子功能失活， 2) 测量转录率的变化作为函数 特定 FMR1 启动子元件和 CGG 重复序列的甲基化状态 尺寸， 3）通过CGG重复评估转录作为 体内和体外三联体扩增的程度， 4) 使用以下方法绘制与 RNA 密切接触的 FMRP 残基图 纯化 FMRP 和修饰 RNA 的光交联。 这将 补充 FMR 基因产物的高分辨率结构研究。 将测试 FMR 蛋白 RNA 接触表面的突变体 用于体外和体内功能。