CCR4-NOT新成员的鉴定及其在m6A介导的RNA降解中的作用

腺苷酸N6位甲基化(N6-methyladenosine,m6A)是真核生物mRNA和长非编码RNA上最常见的内部修饰，也是最先被发现能够调控基因表达、参与多种重要生理过程的RNA修饰。YTHDF家族蛋白可以识别结合RNA上的m6A，影响所在RNA的翻译或降解。申请人已发表的研究证明YTHDF2通过直接相互作用招募脱腺苷酸酶复合体CCR4-NOT，促进RNA的脱腺苷酸化与降解。然而m6A修饰引起RNA降解这一过程的选择性与调控机制仍属未知。为了理解其中机制，我们运用质谱寻找CCR4-NOT的新成员，鉴定得到了RNF219。本项目计划首先阐明RNF219与CCR4-NOT的相互作用，然后运用瞬时诱导的RNA表达系统，研究RNF219对m6A RNA脱尾降解的作用与机制，并且用高通量测序找出RNF219的靶基因，探索RNF219对干细胞功能的影响，为深入理解m6A修饰的生物学功能提供理论基础。

The methylation at N6 position of adenosine (m6A) is the most abundant RNA modification within protein-coding and long noncoding RNAs in higher eukaryotes. m6A is also the first RNA modification discovered to regulate gene expression and participate in a variety of critical biological processes. YTHDF proteins are the readers of m6A, the binding of which results in the alteration of the translation efficiency or stability of m6A-containing RNAs. Our published work demonstrate that YTHDF2 directly recruits the CCR4-NOT deadenylase complex to promote the deadenylation and decay of m6A-containing RNAs. However, the selectivity and regulation of m6A-mediated RNA decay still remain elusive. To further investigate the mechanism, we searched for novel regulators of CCR4-NOT complex by Mass-spectrum and found RNF219. In this study, we will first characterize the interaction between RNF219 and CCR4-NOT, and then take advantage of a well-established RNA decay monitor system to investigate the effect and mechanism of RNF219 in m6A-mediated RNA decay. In addition, we will identify the target genes of RNF219 in embryonic stem cells by RNA sequencing analysis and explore the function of RNF219 in early development.