m6A RNA 甲基化依赖的RNA二级结构改变对RNA结合蛋白和小RNA特异性靶向的调控

m6A (N6-methyladenosine) RNA methylation is a widespread and reversible internal modification on the mRNA of Eukaryotes. m6A tends to form single-stranded RNA, and RNA binding proteins (RBP) usually bind their targets with specific secondary structures. Our pilot study showed that many RNA binding proteins had significant enrichment or depletion of binding sites on m6A regions. Therefore, we propose the following hypothesis: m6A dependent regulation of RBP targeting through structure switches is a common and important mechanism in biology. On the other hand, since miRNAs bind to their targets based on base pairings, which form double-stranded RNAs, we propose that m6A dependent structure switches also regulate the targeting and functions of miRNAs. In this project, we will take advantage of the technologies of genome-wide profiling of RNA secondary structures to identify the switches of RNA secondary structures due to knocking out the m6A methyltransferase METTL3. We will then analyze the effects of these structure switches on the targeting and functions of RBPs and miRNAs followed by experimental validations. We will finally have a clear answer for the question whether it is a common function of m6A to regulate the targeting of RBPs and miRNAs. We will further elucidate the functions of m6A and provide a new prospective on understanding the mechanisms of RBPs and miRNAs to recognize their targets specifically.

m6A RNA甲基化是真核细胞mRNA上广泛存在的的可逆的非末端修饰。m6A偏好形成単链RNA，而RNA结合蛋白通常也对靶点的二级结构有偏好。我们前期研究发现很多RNA结合蛋白的结合位点在m6A位点显著富集或缺乏。由此，我们提出m6A通过影响RNA二级结构参与RNA结合蛋白对靶点的特异性识别是一个广泛的机制并有着重要的生物学意义。由于小RNA与靶点的结合后也形成双链，因此我们大胆推测m6A也参与调控miRNA对靶点的识别并影响其功能。本项目拟采用RNA二级结构测序技术系统地研究敲除m6A甲基转移酶METTL3后RNA二级结构的变化，并进一步分析和验证这些变化对特定RNA结合蛋白以及miRNA的靶点及功能的影响。旨在回答m6A是否能够广泛影响RNA结合蛋白以及miRNA 的结合靶点。本项目将进一步阐明m6A的功能并为理解RNA结合蛋白和miRNA特异性识别靶基因的机制提供崭新的视角。

m6A RNA修饰是真核生物mRNA和长非编码RNA上最丰富的非末端修饰，其在各种生理和病理过程中都发挥了重要作用。然而，并不清楚m6A如何实现特异性调控并形成细胞特异的修饰模式。为了回答这个问题，我们建立了一套基于m6A共甲基化网络的计算方法来系统地鉴定细胞特异的m6A反式调控因子，揭示了大量RNA结合蛋白很可能通过特异性调控m6A参与建立细胞特异的m6A甲基化模式。进一步通过上述共甲基化网络模块的分析预测了一个潜在的m6A调控因子SRSF7很可能参与调控与胶质瘤相关的m6A位点，并发现SRSF7通过招募甲基转移酶复合物选择性地促进胶质瘤细胞中一小部分m6A位点的修饰，这些被修饰细胞的位点能够特异地调控胶质瘤细胞的增殖和迁移从而促进胶质瘤的发展。此外，我们发现假基因尤其是加工型假基因的RNA在灵长类进化中在达尔文正向选择作用下快速积累形成m6A基序的突变，导致m6A水平不断上升，从而通过m6A修饰在胞质降解这些假基因。最后，我们开发了一种基于ABE单碱基编辑的方法在全转录组水平系统地筛选出76个促进以及137个抑制人胚胎干细胞向内胚层分化的m6A位点，进一步阐明了SOX2以及两个未知的内胚层分化相关基因SDHAF1和ADM的m6A位点通过YTHDF2调控胚胎干细胞向内胚层分化的作用和机理。我们根据领域前沿的发展形势，及时对项目的研究目标做出了调整，并顺利实施。本项目共在Nucleic Acids Research (2篇), Genome Biology, Nature Communications, Genomics, Proteomics & Bioinformatics 杂志发表5篇SCI论文，这5篇论文均为中科院分区一区论文，且项目负责人均为通讯作者（含共同）。项目成果从多个角度揭示了m6A RNA修饰形成特异性修饰的机理和功能，并且揭示了其在细胞命运决定中的作用，为深入理解m6A的调控机理和意义奠定了基础。