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

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.

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。

哺乳动物中RNA修饰m6A在脑组织中的丰度最高，提示其在神经系统中发挥重要的生物学作用。基于脑组织基因表达调控的时空特异性，我们首先利用C57/BL6小鼠比较了不同脑区的RNA甲基化水平；其次以大脑皮层与小脑为研究对象，比较了m6A调控因子的表达水平；而后通过m6A-seq比较二者之间的甲基化差异。发现小脑的甲基化水平与甲基化RNA的数量均高于大脑皮层，而且其甲基化位点的分布亦有不同之处。脑组织中有多种神经细胞，通过生信分析表明神经元里的RNA甲基化程度要高于胶质细胞；同时发现FMRP蛋白结合的mRNA中多数均含有m6A，提示FMRP是一种新的m6A结合蛋白，而m6A可能参与调控FMRP结合RNA。.而后我们进一步深入研究m6A在小鼠小脑发育过程中的功能。通过在整体水平以及表观转录组水平上分别对野生型小鼠小脑发育过程中四个时间点的甲基化分析，发现m6A甲基化与去甲基化的动态过程贯穿于小脑出生后发育的整个过程，而以小脑发育早期最为活跃。同时发现小脑发育四个阶段存在各自特异的甲基化RNA，其甲基化水平与甲基化位点分布的表现各不相同，并且功能聚类分析表明这些特异甲基化的产生很有可能与不同的小脑发育阶段功能需求密切相关。与动态的甲基化水平相呼应，m6A相关蛋白在四个时间点的表达也呈现典型的时空特异性表达模式。为证实m6A参与小脑发育调控，该研究利用慢病毒系统分别将甲基化酶METTL3在小鼠小脑内进行敲低和过表达，并发现因此导致的小脑发育障碍。进而利用去甲基化酶Alkbh5基因敲除小鼠，发现在低压低氧环境下ALKBH5缺失导致小鼠小脑发育缺陷；与野生型小鼠比较发现Alkbh5缺失引起了小鼠小脑RNA m6A甲基化谱严重紊乱，并且进一步研究发现部分甲基化失衡会造成小脑发育相关的RNA出核异常。.总之，我们率先证明了RNA甲基化调控在脑组织的时空特异性；首次发现FMRP与m6A结合；首次绘制了正常小鼠小脑出生后发育过程中的RNA m6A甲基化谱以及m6A相关蛋白的表达谱，同时首次揭示了RNA m6A甲基化平衡对于维持小鼠小脑发育的必要性，为研究神经系统发育的机制提供了新思路。