Dynamic regulation of translation by fragile X mental retardation protein

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DESCRIPTION (provided by applicant): The fragile X mental retardation protein FMRP binds ~4% of brain mRNAs but it is still unknown how FMRP regulates their translation. The preliminary data demonstrate association of the putative RNA helicase MOV10 with FMRP. The objective of the proposed research is to understand how MOV10 interacts with FMRP to regulate translation of its associated mRNAs. The hypothesis is that MOV10 regulates translation of FMRP-bound mRNAs by unfolding secondary structures in the RNA. The rationale for the proposed research is that understanding how MOV10 functions will give insight into translation regulation, including virus production and the microRNA pathway. MOV10 also activates translation of the FMR1 mRNA, which could lead to novel treatment strategies. Specific Aim 1 tests the hypothesis that MOV10 unwinds secondary structures in the 5' untranslated region (UTR) to facilitate translation initiation. Methods: Use single-molecule FRET to examine whether MOV10 unfolds RNAs, use translation assays to examine whether MOV10 activates translation and cell culture assays to examine how FMRP recruits MOV10. Specific Aim 2 tests the hypothesis that MOV10 suppresses translation of some FMRP-associated mRNAs by unwinding structures in the 3'UTR to expose target sequences for miRNA-mediated suppression. Methods: examine the miRNA- dependence of MOV10-FMRP regulation of PSD-95 and NR2A luciferase reporters using the methods described in Specific Aim1. Specific Aim 3 tests the hypothesis that the mRNAs in the FMRP-MOV10 complex are the FMRP-associated mRNAs that are regulated at the level of translation by MOV10. Methods: isolate the shared RNAs using serial immunoprecipitation to capture FMRP-MOV10-RNA complexes from brain and identify the captured RNAs by deep sequencing, as well as the specific region of the RNA bound by MOV10 using HITS-CLIP. The contribution of the proposed research is to understand how translation of FMRP-associated mRNAs is regulated. It is innovative because it uses single-molecule approaches to characterize a new helicase and focuses on a novel approach for understanding how FMRP regulates translation of its bound mRNAs through MOV10.