基于上下文的RNA甲基化(m6A)谱的比较分析

The m6A methylation is the most widespread type of RNA modification to date. It could be involved in multiple layers of gene expression regulations including but not limited to RNA degradation, translation and alternative splicing. Intuitively, the functional readout of the m6A modification in physiological and pathological processes depends on its context on the target transcripts, as the functional elements in the context are often crucial for the interplay between m6A modification and other regulatory factors. Thanks to the rapid accumulation of m6A methylation profile data, in this project, we could perform a comprehensive computational analysis to compare m6A methylation across different cell types with specific consideration of the context (e.g. microRNA target sites) similarity between modification sites. We will first build an online m6A modification site context visualization tool to explore the functional elements in the context. Moreover, a context similarity-regularized clustering algorithm will be applied to the comprehensive methylation dataset, in order to identify cell specific functional module of m6A modification. Finally, because our previous work has shown that the m6A modification is likely to play a role in the proliferation and migration of vascular smooth muscle cells (VSMCs). Therefore, we will obtain the m6A methylation profile of VSMCs by MeRIP-seq and apply our clustering method to predict the VSMC-specific m6A target genes that could play important roles in VSMC proliferation and migration. A preliminary experimental validation of our prediction will also be performed. In all, our work will not only establish useful tools for analyzing and comparing the functional context features of m6A methylation profiles, but also provide clues for understanding the intrinsic modularity organization of m6A modification.

RNA m6A 甲基化修饰是已知最普遍的RNA转录后修饰类型，它参与到RNA降解、翻译、可变剪接等重要的基因表达调控机制。多项证据表明，m6A修饰在生理与疾病过程中的作用依赖于m6A靶基因修饰位点上下文的功能元件及与其它因子的相互作用。得益于高通量m6A甲基化谱数据的快速积累，本项目拟结合多种功能上下文注释信息（如microRNA靶位点等），综合比较不同细胞中的m6A甲基化谱。首先构建一个m6A修饰位点上下文的在线可视化工具。然后，采用一种带有上下文特征相似性约束的聚类方法，鉴定细胞特异的m6A位点的功能模块。最后，结合前期工作对m6A在血管平滑肌细胞增殖与迁移表型转化过程中的功能研究，测定血管平滑肌细胞的m6A甲基化谱，并采用上述聚类方法预测这一过程中的关键m6A靶基因并进行初步实验验证。本项目的实施将不仅为m6A甲基化的计算分析提供工具，还将有助于理解甲基化谱内在的调控模块结构。

RNA m6A 甲基化修饰广泛参与到RNA降解、翻译、可变剪接等转录后基因表达调控，而越来越多的实验证据表明，m6A修饰在生理与疾病过程中发挥的调控作用依赖于m6A靶基因修饰功能位点上下文的功能元件及其与其它调控因子的相互作用。本项目提出结合功能上下文注释信息，建立新的m6A甲基化谱的计算分析方法。首先，我们揭示MeRIP-Seq测定的m6A甲基化谱存在内在实验室偏倚，并提出对应的m6A甲基化谱校正方法m6Acorr。开发Nm核糖甲基化、ac4C乙酰化、RNA糖基化等RNA修饰位点预测工具，并挖掘糖基化位点相关的功能上下文特征。然后，通过大规模CLIP-Seq数据重分析，筛选影响mRNA和蛋白水平基因表达差异的关键RNA结合蛋白。采用图正则化的非负矩阵分解方法，将RNA结合蛋白相关的功能上下文特征以图正则项的形式整合，建立基于上下文特征的m6A甲基化谱比较分析工具m6Adecom。最后，与实验团队合作，发现METTL3催化的m6A甲基化参与血管平滑肌细胞表型转换和巨噬细胞极化调控，分析血管平滑肌细胞和巨噬细胞的m6A甲基化谱，初步鉴定了以上过程中关键的m6A靶基因KLF4和 STAT1。本项目不仅为如何整合上下文特征分析m6A甲基化谱提出了有效的策略，还为理解m6A甲基化参与血管平滑肌表型转化、巨噬细胞极化等重要生理或病生理过程的机制提供新的线索。