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.
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