RNA m6A甲基化动态修饰机制和动态结构变化的研究

RNA m6A methylation is an important post-transcriptional modification, which is widely present and is involved in multitude of physiological and pathological processes. A protein machinery consisting of METTL3-METTL14 methyltransferase and other cofactors is responsible for RNA m6A methylation. Previously, we have determined the crystal structure of the catalytic domain of METTL3-METTL14. Our more recent experimental data also found that METTL3 contains a zinc finger domain, which specifically binds to RNA substrate and plays a key role for enzymatic catalysis. This project will characterize the dynamic structure of core methyltransferase in the RNA modification processes, and to reveal the regulatory mechanism by cofactor proteins and environmental factors. On the other hand, this project attempts to analyze the effect of methylation for RNA regarding its ensemble structure and function, and to establish the relationship between RNA methylation and its sequence and structure. To this end, the project will integrate a variety of steady-state characterization and dynamic analysis techniques, and use conjointly single-molecule data and bulk measurements, with additional means of bio-conjugation. Together, the project intends to reveal the physical chemical nature for dynamic modifications and conformational dynamics of biological macromolecules. Two major applicants in this project have a wealth of relevant research experience and long-term collaboration, and are complementary in their expertise. The implementation of this project will account for the dynamic modification and regulation mechanism of RNA m6A modification, and will provide theoretical guidance for future development of relevant chemical interventions.

m6A甲基化是一种重要的RNA转录后修饰，分布广泛，在生命过程中的重要性日益凸显。RNA m6A甲基化修饰是METTL3-METTL14甲基转移酶及辅助因子所组成的蛋白质机器介导。我们已解析了其催化结构域的结构，预实验还发现METTL3中的锌指结构域能特异性结合RNA底物，对酶活起关键作用。本项目拟表征核心甲基化酶在RNA动态修饰过程中的结构变化，揭示辅助因子和环境因素对动态结构和催化过程的调控。另一方面，本项目拟分析甲基化动态修饰对RNA结构和功能的影响，确立甲基化修饰与序列、结构的关联。为此，本项目将集成多种稳态表征和动态分析技术，整合单分子测定和系综平均数据，并借助化学标记手段，发现生物大分子动态修饰和动态变化的物理化学本质。两名项目主要申请人有丰富的相关研究经验，并长期合作、技术互补。本项目的实施将阐释RNA m6A甲基化的动态修饰和调控的机制，为发展相应的化学干预提供理论指导。

RNA m6A甲基化修饰在生命活动中扮演着重要的调控作用。对RNA m6A甲基化动态修饰机制和动态结构变化的研究能够为发展相应的化学干预手段提供帮助。围绕研究目标，项目研究团队取得了一系列的成果和进展：通过解析甲基化酶锌指结构域（ZFD）的溶液结构，阐明了m6A甲基化酶复合物识别底物RNA的分子机制；以METTL3-METTL14甲基化酶复合物为核心，成功组装了包含不同辅助因子蛋白的四元、五元和六元蛋白质机器复合物，并将进一步利用冷冻电镜解析这些复合物的结构，揭示甲基化动态修饰的机制；发展了溶液顺磁核磁研究蛋白质与RNA的结构和动态变化的新方法，为生物大分子动态修饰的研究提供了一个有力的工具；发展了整合溶液核磁共振，小角散射，荧光和分子动力学模拟表征RNA动态结构的新方法；发展了在细胞水平研究蛋白质结构与相互作用新方法，能够为进一步在细胞水平研究生物大分子动态修饰提供帮助。在本项目的资助下，共发表研究论文7篇，另有多篇论文在撰写和投稿中，获得专利授权1项，培养出站博士后2人，培养毕业博士研究生5人，培养毕业硕士研究生2人；建立了研究生物大分子动态修饰的研究平台。