FMRP通过miRNA途径介导翻译抑制的分子机制研究

microRNAs (miRNAs) play an important role in translational regulation and are involved in almost all physiological and pathological processes. Fragile X syndrome (FXS) is the first disease that has been linked to the dysfunction of the miRNA pathway thus far. Fragile X mental retardation protein (FMRP) is the key pathogenic factor, which selectively binds to a large number of mRNAs and negatively regulates their translation. Recent studies have shown that FMRP can inhibit translation through miRNA pathway. However, it remains unclear about the molecular mechanism of FMRP associating and regulating miRNA-induced translational repression. Base on the sequence analysis of the target mRNAs of MRP associated miRNAs, this project aim to study the linkage between the regulation of the specific miRNA pathway by FMRP and the targeting of the same mRNA by FMRP in the neighborhood of the miRNA recognition element . To a further step, by the use of single-molecule fluorescence detection, we try to reveal the molecular mechanism involved in the interaction between FMRP and miRNA-induced silencing complex (miRISC). The achievements of this project may provide new insights into the molecular mechanism of miRNA-mediated translational regulation and pathogenesis of FXS, providing a theoretical basis for accurate diagnosis and effective drug design and treatment of the disease.

miRNA在翻译调控过程中起着重要的作用，几乎参与到所有的生理与病理进程。脆性X染色体综合症（FXS）是首个与miRNA沉默途径建立联系的遗传性智力低下疾病。脆性X智力迟钝蛋白（FMRP）是致病的关键，它选择性的与众多mRNA结合并抑制其翻译。近来研究发现FMRP能够通过miRNA介导翻译抑制，但是FMRP关联和调控miRNA介导的翻译途径的分子机制还不清楚。本项目基于对FMRP选择性关联的miRNA的靶mRNA的序列分析，计划研究FMRP调控特定的miRNA沉默途径与FMRP在miRNA识别序列邻近区域靶向共同靶mRNA的联系，进一步通过单分子荧光检测新方法揭示FMRP调控miRNA沉默复合体（miRISC）的分子机制。本项目研究成果有望为了解miRNA介导的翻译调控机制和FXS的分子致病机理提供新的视野，从而为疾病的精确诊断以及有效的药物设计和治疗提供理论依据。

本项目对FMRP关联和调控miRNA介导的翻译途径的分子机制进行了研究。以四组代表性miRNA和靶蛋白为研究对象，验证了FMRP调控特定miRNA沉默途径依赖于FMRP靶向共同的靶mRNA中miRNA识别位点的邻近区域，并且FMRP需要结合到miRNA识别序列的下游邻近区域才能发挥调控作用（ “下游调控模式”）。以受FMRP调控的miR196a介导的HOXB8翻译抑制为例，研究发现 FMRP与靶mRNA的亲和力不受到FMRP磷酸化状态的影响。基于序列和蛋白结构分析，结合突变验证证明FMRP通过与AGO2的MID结构域内T544-K533-K570结合口袋相互作用影响miRNA沉默效果。进一步的，本项目还详细研究FMRP调控特定的miR-196a-HXOB8沉默途径时FMRP、AGO2、miR196a和靶mRNA这四者间动态的相互作用过程。比较了FMRP磷酸化或去磷酸化时FMRP与AGO2之间FRET效率的分布和波动差异，发现磷酸化的FMRP能够促进miR196a介导的HOXB8翻译抑制的作用是因磷酸化能增大FMRP与AGO2的结合和解离的时间常数，从而能延长miRNA沉默复合体在mRNA上停留的时间。进一步，更为细致的分析比较在没有或者存在FMRP作用下AGO2-miRNA复合体从游离到结合mRNA过程中AGO2的构象变化，发现FMRP与AGO2的相互作用能增加AGO2蛋白的N端与C端的间距，这可能是FMRP能促进AGO2与靶mRNA结合的稳定性的原因所在。