RNA m6A 修饰甲基转移酶复合物及其表观调控因子鉴定和作用机制研究

In messenger RNA (mRNA) methylation is mainly found on N6-adenosine generating N6-methyl-adenosine (m6A), but how it regulates mRNA metabolism remains largely unknown. The applicant identified for the first time the m6A methyltransferase tricomplex METTL3/METTL14/WTAP catalyzing the m6A formation and the only two known m6A demethylases FTO and ALKBH5 that via an oxidative reaction removes the methylgroup on m6A. The dynamic regulation of m6A methylation is important for energy metabolism and spermatogenesis. The discovery of the RNA m6A methyltransferases and the demethylases demonstrate that besides the vital role of DNA and histone methylation, m6A methylation on RNA also plays essential roles in epigenetic regulation of basic life processes in mammals and is now considered a novel epitranscriptomic marker of profound biological significance. The regulation of m6A formation and maintenance and its regulatory roles in mRNA metabolism has therefore now become a new frontier in RNA biology research. .In our new preliminary studies, we screened 35 potential candidates interacting with the m6A methyltransferase tricomplex, and found that METTL3 co-localizes with several histone modifications and that non-coding microRNA regulates cellular mRNA m6A modification levels. These results suggest that epi-regulatory factors such as histone modifications and microRNA may regulate the m6A methyltransferase and modulate m6A level. By integrating multi-cross-disciplinary omics technologies including epi-modifications, proteomics, genomics, etc., this project aims at identifying new enzymatic components of the m6A methyltransferase complex as well as epi-regulatory factors including histone modifications and microRNA and determine their function in mRNA metabolism and potential association with human diseases. Unveiling of the molecular mechanisms of m6A formation and maintenance will provide compelling evidences directly supporting the m6A-modified RRACH (R=G,A; H=A,C,U) sequences in mRNA as new cis-regulatory elements, and provide new important insights into the mechanistics of RNA transcriptomics..

6甲基腺嘌呤（m6A）是mRNA主要修饰形式,但其调控mRNA代谢功能未知。其甲基转移酶复合物METTL3/METTL14/WTAP和去甲基化酶FTO和ALKBH5的发现，首次证实m6A修饰的可逆性，提示m6A调控mRNA代谢。m6A修饰形成维持机制及其调控mRNA代谢，已成为RNA甲基化表观转录组学研究新领域的重要科学问题。在前期研究中，申请人筛选到35个与m6A甲基转移酶三聚体互作候选蛋白，发现METTL3与多种组蛋白修饰共定位及非编码microRNA调控m6A修饰水平，提示这类表观因子调控m6A甲基转移酶。本项目整合多组学(表观修饰、蛋白组、基因组)等交叉学科技术，鉴定m6A甲基转移酶全酶组分及其表观调控因子组蛋白修饰和microRNA，解析m6A与mRNA代谢和疾病关联，阐明m6A修饰形成维持机制，为m6A修饰作为mRNA新顺式调控元件和揭示RNA表观转录组学核心规律提供理论依据。

RNA m6A甲基化修饰调控规律和功能研究，为mRNA加工代谢和生命活动增加了一种新的甲基化顺式调控机制。本项目重点揭示m6A位点形成的分子机理，阐明m6A调节靶基因mRNA加工和代谢及功能分子机制，探讨m6A修饰与生物学功能关联。研究首先揭示了RNA m6A甲基化位点选择性形成的具体分子机制。团队合作发现microRNA的靶点与m6A修饰存在很好的共定位现象，并验证了miRNA可以通过序列互补机制调控m6A修饰。敲低miRNA或其加工酶Dicer显著地降低细胞水平m6A修饰的丰度，相反过表达Dicer则增加了m6A修饰的丰度。Dicer调控m6A甲基转移酶催化亚基METTL3在细胞核内核小斑的定位。Dicer和microRNA均可以调控METTL3结合mRNA的亲和力。这些结果表明非编码microRNA能够调控RNA m6A甲基转移酶选择底物mRNA的甲基化位点。研究进一步证实了m6A对mRNA加工和代谢的调节机制，明确了m6A调控mRNA剪接，翻译及转录终止的分子机理。YTHDC1可促进SRSF3，同时抑制SRSF10在细胞核内的核小斑定位和mRNA结合能力，从而调控m6A修饰外显子的选择性剪接。此外，发现m6A结合蛋白YTHDF3和YTHDF1协同作用促进m6A修饰mRNA的翻译过程。同时，研究发现m6A甲基化调控R-loop形成及转录终止。同时，研究揭示了m6A在中枢神经系统发育中的功能，明确了m6A调控小脑发育，少突胶质细胞特化和髓鞘化的分子机理。Mettl3敲除引起的m6A甲基化修饰缺失严重影响小脑发育。研究发现新的m6A结合蛋白Prrc2a调控少突胶质细胞的特化和髓鞘化。综上所述，此项目研究阐明了micoRNA调控m6A修饰选择性形成的分子机理，揭示了m6A修饰对mRNA加工和代谢调节机制，发现m6A修饰调控中枢神经系统发育等重要生物学功能。