RNA m6A甲基化修饰调控mRNA选择性剪接机制研究

mRNA alternative splicing increases the proteomic diversity. It is regulated by cis-regulatory element and trans-acting splicing factors, and can also be modulated by epigenetic modification, such as DNA methylation and histone modifications. It has become a hot topic in the field of RNA biology. The applicants previously together with other groups identified and characterized the mRNA methylation N6 methyladenosine (m6A) methyltransferase core complex; METTL3/METTL14/WTAP. Interference with the m6a methyltransferase results in changes in gene expression and mRNA splicing and we have primary data suggesting that the m6A modification may serve as a new cis-regulatory element participating in the regulation of mRNA alternative splicing. In previous studies, we discovered that m6A can regulate the process of pre-adipocyte differentiation, mRNA alternative splicing, and the association of important mRNA splicing factors with mRNA. In this project, we place particular emphasis on obtaining a thorough mechanism understanding of the relationship between m6A and alternative splicing. We have established high-throughput RNA-seq and m6A-IP-seq technology platforms, and developed appropriate bioinformatics for data analyses. This in combination with state-of-the-art molecular biology techniques provide us with the necessary tools required for elucidating how m6A influence splice site selection by splicing factors, an essential event in the regulation of alternative splicing, and provide a base for studying m6A as a potential novel mRNA cis-regulatory element important for alternative splicing.

mRNA选择性剪接增加了蛋白质组多样性，受到顺式元件和反式因子调控，还受到表观修饰如DNA甲基化、组蛋白修饰调控，是RNA生物学研究热点。申请人首次发现了mRNA主要甲基化形式6甲基腺嘌呤(m6A)修饰甲基转移酶三聚体METTL3/METTL14/WTAP，其酶活性缺失导致参与调控mRNA代谢重要因子剪接模式变化，提示mRNA m6A修饰可能作为一种新的顺式元件，参与调控mRNA选择性剪接。 在前期研究中，发现m6A修饰调控脂肪前体细胞分化、基因选择性剪接，及与重要剪接因子结合mRNA的位点和亲和力密切关联。本项目结合分子细胞、高通量m6A-IP-Seq和转录组测序和生物信息学多学科交叉技术，探索m6A修饰与mRNA反式剪接因子介导选择性剪接及靶位点结合之间的关联，重点阐明m6A修饰调控mRNA 选择性剪接作用机制，为研究m6A修饰作为mRNA选择性剪接进程中新的顺式调控元件提供理论依据。

首先揭示了FTO可调节多转录本基因的表达水平和mRNA的剪接形式。分化各时期的m6A修饰以及FTO介导下的m6A修饰在5’和3’剪接位点相邻的外显子区域显著富集。并且这些m6A修饰位点与mRNA剪接调节因子SRSF1、SRSF2识别的剪接增强元件（ESE）结合序列具有空间位置重叠关系，而SRSF3和SRSF4，以及hnRNPC/H的结合序列却不存在该种现象。FTO缺失引起的靶基因转录本m6A水平升高，可增加SRSF2蛋白对RNA的结合能力，进而提高基因特定外显子的保留水平。而METTL3缺失可导致相反结果。.我们同时揭示了m6A调控选择性剪接的具体机制。结合免疫共沉淀技术和二级串联质谱技术发现了5个与m6A核内结合蛋白YTHDC1相互作用的SR家族蛋白SRSF1、SRSF3、SRSF7、SRSF9、SRSF10。通过在转录水平上的剪接分析，我们发现YTHDC1、SRSF3以及METTL3能促进外显子保留而SRSF10则促进外显子剪接。为了探究YTHDC1和SR家族蛋白靶向外显子的剪接模式，结合光活性增强的核糖核苷交联和免疫共沉淀测序（PAR-CLIP-seq）以及转录组测序（RNA-seq）数据，我们分析发现YTHDC1和SRSF3促进它们靶向外显子的保留而SRSF10则促进它靶向外显子的切除，而其它SR蛋白并没有显著的偏好性。通过免疫共沉降技术，我们分别在体内和体外证明了YTHDC1与SRSF3和SRSF10具有直接相互作用，并且SRSF3能与SRSF10竞争性的结合YTHDC1。它们竞争性的结合YTHDC1进一步印证了SRSF3和SRSF10相反的剪接模式。通过PAR-CLIP技术结合3’末端生物素标记实验，我们发现当敲低YTHDC1后，SRSF3对RNA的结合信号减弱而SRSF10对RNA的结合信号增强。这说明YTHDC1能招募SRSF3同时抑制SRSF10结合在RNA上，从而导致剪接形式的改变。通过外源回补实验，我们进一步发现野生型的YTHDC1能够回补因敲低YTHDC1引起的SRSF3和SRSF10对RNA的结合能力以及选择性剪接紊乱，但是m6A结合关键氨基酸突变的YTHDC1则不能回补。这说明YTHDC1介导的剪接变化是m6A依赖的。我们的研究为细胞核内m6A读码器YTHDC1影响前体mRNA剪接因子与mRNA结合的选择性剪接调控的机制提供了有力的证据。