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基因功能的丧失会导致脆性X染色体