Dynamic regulation of translation by fragile X mental retardation protein

描述（由申请人提供）：脆性X智力迟钝蛋白FMRP结合约4%的脑mrna，但FMRP如何调节其翻译仍不清楚。初步数据表明，假定的RNA解旋酶MOV10与FMRP有关。本研究的目的是了解MOV10如何与FMRP相互作用以调节其相关mrna的翻译。假设是MOV10通过展开RNA中的二级结构来调节fmrp结合mrna的翻译。这项拟议研究的基本原理是，了解MOV10的功能将有助于深入了解翻译调控，包括病毒产生和microRNA途径。MOV10还激活FMR1 mRNA的翻译，这可能导致新的治疗策略。特异性目标1验证了MOV10解开5'非翻译区（UTR）的二级结构以促进翻译起始的假设。方法：用单分子FRET检测MOV10是否展开rna，用翻译法检测MOV10是否激活翻译，用细胞培养法检测FMRP如何招募MOV10。特异性目的2验证了MOV10通过解开3'UTR中的结构来暴露靶序列以进行mirna介导的抑制，从而抑制某些fmrp相关mrna的翻译的假设。方法：使用Specific Aim1中描述的方法检测MOV10-FMRP调控PSD-95和NR2A荧光素酶报告基因的miRNA依赖性。特异性目的3验证了FMRP-MOV10复合体中的mrna是在翻译水平上受MOV10调控的fmrp相关mrna的假设。方法：采用连续免疫沉淀法从脑内捕获FMRP-MOV10-RNA复合物，分离共享RNA，并通过深度测序对捕获的RNA进行鉴定，同时利用HITS-CLIP对MOV10结合的RNA的特定区域进行鉴定。本研究的贡献在于了解fmrp相关mrna的翻译是如何被调控的。这是一项创新，因为它使用单分子方法来表征一种新的解旋酶，并专注于一种新的方法来理解FMRP如何通过MOV10调节其结合的mrna的翻译。

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.