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结合了大约4%的大脑mRNAs，但FMRP如何调控它们的翻译仍不清楚。初步数据表明，推测的RNA解旋酶MOV10与FMRP有关。拟议研究的目的是了解MOV10如何与FMRP相互作用来调节其相关mRNAs的翻译。假设MOV10通过展开RNA中的二级结构来调节FMRP结合mRNAs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，包括病毒生产和microRNA途径。MOV10还激活了FMR1mRNA的翻译，这可能导致新的治疗策略。特定目的1检验了MOV10在5‘非翻译区(UTR)解压二级结构以促进翻译起始的假设。方法：用单分子FRET检测MOV10是否打开RNA，用翻译实验检测MOV10是否激活翻译，用细胞培养实验检测FMRP如何招募MOV10。特异性目标2验证了这样的假设，即MOV10通过解开3‘UTR中的结构来揭示miRNA介导的抑制靶序列，从而抑制一些FMRP相关mRNAs的翻译。方法：采用特异性Aim1中描述的方法，检测Mov10-FMRP对PSD-95和NR2A型荧光素酶报告分子的miRNA依赖性调节。特定目的3验证了这样的假设，即FMRP-MOV10复合体中的mRNAs是FMRP相关的mRNAs，由MOV10在翻译水平上进行调节。方法：用连续免疫沉淀法分离共享RNA，捕获FMRP-MOV10-RNA复合体，对捕获的RNA进行深度测序，并用HITS-CLIP方法鉴定与MOV10结合的RNA的特异区。拟议的研究的贡献是了解FMRP相关的mRNAs的翻译是如何受到调控的。它的创新之处在于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mRNAs的翻译。 公共卫生相关性：这项拟议的研究与公共健康相关，因为它探索了最常见的遗传性智力低下形式--脆性X综合征的分子基础，该综合征与自闭症具有共同的神经生物学。了解FMRP结合的RNA是如何在翻译水平上受到调控的，将有助于深入了解新的治疗策略。因此，拟议的研究与NIH任务的一部分有关，即发展基础知识以帮助减轻人类残疾的负担。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it explores the molecular basis of the most common form of inherited mental retardation, fragile X syndrome, which has shared neurobiology with autism. Understanding how the RNAs bound by FMRP are regulated at the translational level will give insight into new therapeutic strategies. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge to help reduce the burdens of human disability.