IDENTIFICATION OF FMRP TARGET RNAS

描述：(改编自申请人的描述)本申请 建议检验脆性X型智力低下是由 FMRP未能结合序列特定的RNA靶标，并未能识别这些靶标 目标RNA。这一问题的三个方面将得到明确解决。 首先，FMRP序列特异的RNA靶标将在体外使用 RNA选择方法，特别是描绘与全长结合的RNA FMRP和FMRP Kh2 RNA结合域，其中I到N突变是 与脆性X智力低下有牵连。此外，诱变研究 将提供对核酸和氨基酸残基的洞察 对RNA-蛋白质识别至关重要。第二，调查人员将利用 这些信息用来识别体内候选的FMRP RNA靶点，使用几个 多余的战略。第三，他们将测试这些候选人的新陈代谢 基因定义的FMR缺失小鼠体内的RNA。调查人员将 利用FMR缺失小鼠和BAC转基因技术挽救FMR-1 FMR-1基因缺失的小鼠在Kh2基因上存在特定的突变 序列特定的RNA结合。携带Kh2基因的小鼠的表型检测 点突变将允许研究人员将神经元的某些方面 由于序列特异性RNA结合失败而导致的功能障碍。 此外，突变株中候选RNA的代谢与 野生型小鼠将允许研究人员得出结论，FMRP对 神经元中的特定RNA靶标。

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.