m6A甲基化RNA在小脑神经元细胞增殖与分化过程中的作用与机制研究

项目名称： m6A甲基化RNA在小脑神经元细胞增殖与分化过程中的作用与机制研究

项目编号： No.31471288

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 牛亚梅

作者单位： 中国医学科学院基础医学研究所

项目金额： 80万元

中文摘要： m6A是一种动态的、广泛存在于多物种的RNA表观遗传修饰标记，受甲基化酶、去甲基化酶双重调控，参与机体代谢、发育、生殖等多过程。我们前期工作表明脑中RNA的m6A甲基化水平很高，且在小脑发育过程中呈动态变化，但功能与机制均不详。本课题中我们将利用MeRIP技术富集不同年龄小鼠小脑中的甲基化RNA，分析m6A在小脑神经元细胞增殖分化等过程中的变化规律，找到调控甲基化的作用机理；而后针对CNM1&2两个具有高甲基化水平且在脑组织中高表达的RNA，结合细胞和动物研究其生物学功能、尤其是甲基化介导的对小脑神经元细胞增殖与分化的作用与分子机制；构建基因敲除动物模型，通过表型分析，寻找甲基化RNA功能异常与疾病的关联。本项目是从RNA甲基化表观遗传角度展开的阐明小脑神经元增殖与分化机制的前沿性研究，研究结果将为揭示小脑发生机理、寻找小脑疾病的诊断和治疗靶标提供重要的理论支持。

中文关键词： RNA甲基化；m6A；RNA表观遗传；细胞增殖与分化；小脑神经元

英文摘要： As one of the most prevalent RNA modifications, N-6-methyladenosine (m6A) has been discovered decades ago in a wide variety of organisms. By using methylated RNA immunoprecipitation enrichment and next generation sequencing technology (MeRIP-seq), more than 7000 kinds of mRNA and 300 kinds of ncRNA have been detected of this methylation sites, which most often occur near stop codon and 3'-UTR with conserved motif. It has been proven to be a dynamic RNA epigenetic mark regulated by both methyltransferases and demethylases. M6A may play an important role in adipogenesis, spermatogenesis, and development according to phenotypic analysis of methyltransferases and demethylases-deficient animal models, however its exactbiological functionsand related mechanisms remain largely unknown. M6A is abundant in brain with a dynamic expression pattern throughout brain development, indicating a possible epigenetic regulatory function in brain. Our preliminary data shows that m6A level is correlated well with cerebellar neurogenesis. Thus, the rational of this studyis to clarify how m6A functions in regulation of cerebellar neurogenesis, especially in cerebellar neuronal cells proliferation and differentiation. By using poly (A) RNA isolated from mice cerebellum of different age, MeRIP-seq will be performed firstly to find out the methylated RNAs involved in cerebellar neurogenesis. Combining with RNA-seq data analysis performed in parallel, a global cerebellar neurogenesis-orientated RNA methylation map will be drawn to uncover the relationship between m6A RNA methylation and cerebellar neurogenesis. Two methylated RNAs (CNM1 & 2) have been selected due to their high methylation level and abundance in brain. After determining the correlation of methylation level and cerebellar neurogenesis in vivo, their biological functions will be investigated in detail.Firstly, RNA subcellular localization, the temporal-spatial expression profiles during neuronal cells proliferation and differentiation will be explored in neuroblastoma cells and mice.Secondly, their effect on cerebellar neuronal cells proliferation and differentiation will be examined by altering RNA expression levels or methylation levels.Thirdly, RNA-IP or RNA-pull down will be performed to explore their interacting partners; effect of methylation level on cellular RNA metabolism will also be evaluated to clarify the molecular mechanism involved in regulation of cerebellar neurogenesis by RNA methylation; Furthermore, corresponding knockout mice will be generated and analyzed of their phenotypes to test their biological functions in vivo. Thisis a novel study aiming to uncover the molecular mechanism of cerebellar neurogenesis from the viewpoint of RNA methylation. The results will not only help to understand the importance of RNA epigenetics in various biological procedures, but also provide important information in exploring novel RNA biomarkers and therapeutic targets of cerebellar diseases.

英文关键词： RNA methylatioin;N6-methyl-adenosine;RNA epigenetics;cells proliferation and differentiation;cerebellar neuronal cells