Dynamic regulation of translation by fragile X mental retardation protein

描述（由申请人提供）：脆性X智力低下蛋白FMRP结合约4%的脑mRNA，但仍不清楚FMRP如何调节其翻译。初步数据表明推定的RNA解旋酶MOV 10与FMRP相关。这项研究的目的是了解MOV 10如何与FMRP相互作用，以调节其相关mRNA的翻译。假设是MOV 10通过解折叠RNA中的二级结构来调节FMRP结合mRNA的翻译。这项研究的基本原理是，了解MOV 10的功能将有助于深入了解翻译调控，包括病毒的产生和microRNA途径。MOV 10还激活FMR 1 mRNA的翻译，这可能导致新的治疗策略。特定目的1检验了MOV 10解旋5'非翻译区（UTR）中的二级结构以促进翻译起始的假设。研究方法：使用单分子FRET检测MOV 10是否展开RNA，使用翻译测定检测MOV 10是否激活翻译，使用细胞培养测定检测FMRP如何招募MOV 10。特异性目的2检验了MOV 10通过解旋3 'UTR中的结构以暴露靶序列用于miRNA介导的抑制来抑制一些FMRP相关mRNA的翻译的假设。方法：使用Specific Aim 1中描述的方法检查PSD-95和NR 2A荧光素酶报告基因的MOV 10-FMRP调节的miRNA依赖性。特定目的3检验了FMRP-MOV 10复合物中的mRNA是在翻译水平上受MOV 10调控的FMRP相关mRNA的假设。研究方法：使用系列免疫沉淀分离共享RNA以从脑中捕获FMRP-MOV 10-RNA复合物，并通过深度测序鉴定捕获的RNA，以及使用HITS-CLIP鉴定MOV 10结合的RNA的特定区域。这项研究的贡献是了解FMRP相关mRNA的翻译是如何调节的。它是创新的，因为它使用单分子方法来表征一种新的解旋酶，并专注于了解FMRP如何通过MOV 10调节其结合mRNA的翻译的新方法。

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.