哺乳动物中SR蛋白激酶SRPK1通过调控Drosha的磷酸化参与microRNA生物合成的机制研究

项目名称： 哺乳动物中SR蛋白激酶SRPK1通过调控Drosha的磷酸化参与microRNA生物合成的机制研究

项目编号： No.31500661

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物物理、生化与生物分子学、生物力学与组织工程

项目作者： 王萍萍

作者单位： 山东师范大学

项目金额： 21万元

中文摘要： miRNA是一类广泛存在的非编码小RNA，维持其表达具有重要的意义。参与miRNA合成的Drosha的N端含有RS-rich 功能域，可能是SR蛋白激酶SRPK1的底物。前期的预实验发现，细胞中Flag-SRPK1和v5-Drosha可以免疫共沉淀，证明二者在体内可以互作；miRNA profiling也证明了SRPK1敲除后某些miRNA的合成有显著变化。本课题旨在揭示SRPK1通过调控Drosha磷酸化参与miRNA合成的机制。首先，通过蛋白质互作，磷酸激酶实验以及质谱分析等确认SRPK1可以磷酸化Drosha并找到其磷酸化位点；然后利用定量PCR和Northern blot的方法,分析SRPK1对miRNA合成的影响是否通过Drosha完成；最后，通过免疫荧光分析Drosha的磷酸化变化影响miRNA合成的可能机制。该课题对于理解Drosha表达及miRNA合成的调控方式有重要意义。

中文关键词： SRPK1；Drosha；复合物；非编码RNA生成；蛋白质磷酸化；癌症

英文摘要： miRNAs are a class of short non-coding RNAs that are widely spread in animals and plants. They play key roles in gene regulation, thus it is important to keep their right levels in cells. Drosha, a nuclear RNase III that librates the pre-miRNAs in miRNA biogenesis, contains a RS-rich domain in its N-terminus that might be a substrate of SR protein-specific kinase SRPK1. Our preliminary data showed that Flag-SRPK1 and v5-Drosha can be co-immunoprecipitated in 293T cells, suggesting their interactions in vivo; further experiments by high-throughput miRNA profiling demonstrated that the expression of some miRNAs were strongly affected in the absence of SRPK1. Based on these results, we proposed to use the mouse and human cells as models to explore the potential mechanisms underlying how SRPK1 involves in the miRNA biogenesis through phosphorylating Drosha. First, by using the in vitro protein binding assay together with the kinase assay and mass spectrometry analysis, we are going to confirm whether SRPK1 can phosphorylate Drosha and if it does, where are the critical phosphorylation sites; next, we propose to use qPCR and Northern blots to determine the contributions of SRPK1 to regulating miRNA biogenesis through Drosha based on the results of miRNA profiling; finally, we will investigate the underlying mechanism of SRPK1 regulating the miRNA biogenesis. These studies have the potential to provide mechanistic insights into phosphorylation regulation of Drosha and into the possible mechanism of regulating miRNA biogenesis. We believe the proposed research will have broad implications on cancer biology clinically.

英文关键词： SRPK1;Drosha;microRNA biogenesis;protein phosphorylation;tumorigenesis