IDENTIFICATION OF FMRP TARGET RNAS

描述：（根据申请人的描述改编）本申请 提出测试的假设，脆性X智力低下的结果， FMRP未能结合序列特异性RNA靶点， 靶RNA。 将明确讨论这一问题的三个方面。 首先，FMRP序列特异性RNA靶标将使用体外细胞培养技术鉴定。 RNA选择方法，特别是描绘与全长RNA结合的RNA FMRP和FMRP KH 2 RNA结合结构域，其中I至N突变是 与脆性X染色体智力低下有关 此外，诱变研究 将深入了解核酸和氨基酸残基， 对于RNA蛋白质识别至关重要。 其次，调查人员将利用 使用几种方法，利用这些信息来鉴定候选的体内FMRP RNA靶标， 冗余策略 第三，他们将测试这些候选人的新陈代谢 在遗传定义的FMR缺失小鼠中体内的RNA。 调查人员将 利用FMR敲除小鼠和BAC转基因技术拯救FMR-1 FMR-1基因敲除小鼠，其携带KH 2中的特定突变， 序列特异性RNA结合。 携带KH 2的小鼠的表型检查 点突变体将允许研究人员分配神经元的方面， 由序列特异性RNA结合失败引起的功能障碍。 此外，比较了突变体中候选RNA的代谢， 野生型小鼠将使研究人员得出结论，FMRP作用于 神经元中的特定RNA靶点。

DESCRIPTION: (Adapted from the applicant's description) This application proposes to test the hypothesis that fragile-X mental retardation results from a failure of FMRP to bind sequence-specific RNA targets, and to identify those target RNAs. Three aspects of this issue will be definitively addressed. First, FMRP sequence-specific RNA targets will be identified using in vitro RNA selection methods, specifically delineating RNAs that bind to full-length FMRP and to the FMRP KH2 RNA binding domain, in which an I to N mutation is implicated in fragile-X mental retardation. In addition, mutagenesis studies will yield insight into the nucleic acid and amino acid residues that are critical for RNA-protein recognition. Second, the investigators will utilize this information to identify candidate in vivo FMRP RNA targets, using several redundant strategies. Third, they will test the metabolism of these candidate RNAs in vivo in genetically defined FMR null mice. The investigators will utilize FMR null mice as well BAC transgenic technology to rescue the FMR-1 null mice with FMR-1 genes harboring specific mutations in KH2 necessary for sequence-specific RNA binding. Examination of phenotype of mice harboring KH2 point-mutants will allow the investigators to assign aspects of neuronal dysfunction that result from failures in sequence-specific RNA binding. Moreover, comparison of the metabolism of candidate RNAs in mutant with wild-type mice will allow the investigators to conclude that FMRP acts on specific RNA targets in neurons.