Mechanisms of Regulation of the m6A mRNA Methylation Machinery

项目概要 RNA 修饰（表观转录组）代表转录后基因表达的机制 监管和一个新兴且令人兴奋的生物学领域。 N6-甲基腺苷 (m6A) 是最丰富的后 从酵母到人类，整个真核生物界都检测到了 mRNA 的转录修饰。 m6A 已 涉及多种分子过程，例如 RNA 剪接、RNA 稳定性和 miRNA 加工以及细胞 减数分裂、细胞增殖和胚胎干细胞分化等功能以及疾病状态。的 负责 m6A 的甲基转移酶形成异二聚体，其中 METTL3 提供催化活性。我们 等人发现 METTL3 对增殖、分化和细胞存活具有积极影响。因此，我们 假设 METTL3 代表一个由细胞外和细胞内信号控制的调节中心，允许 细胞通过调节 RNA 代谢来响应发育线索和特定的代谢环境。 尽管我们对 m6A 功能的了解迅速增加，但它还有许多基本方面 至今仍未知的过程。例如，尚待确定甲基转移酶的活性是否为 受上游信号通路调节，如果是这种情况，涉及的机制是什么？ 这种调节对体内平衡和发育的分子和细胞后果？另一个关键问题是 了解如何确定甲基化反应的特异性。我们知道识别序列为 m6A 甲基化由一个非常短的序列基序组成，但尽管该序列在 转录组中只有一小部分此类基序被甲基化。额外的 RNA 结构基序或序列 要求尚未确定。为了回答这些广泛的问题，我们的实验室正在开展一项多学科研究 方法包括分子和细胞生物学、生物化学以及 结构研究辅以新颖的工程小鼠模型。这里描述的程序将使我们能够 解决细胞外刺激下 m6A 甲基化途径的调节机制， 代谢应激和发育。使用最先进的蛋白质组学技术，我们将识别特定背景的后 METTL3 获得的翻译修饰及其对酶活性和特异性的影响。我们会 通过结构技术（包括 X 射线晶体学和冷冻电子显微镜）补充这些研究 定义调控事件对蛋白质复合物形成和底物识别的分子后果。在 同时，我们将使用我们最近开发的小鼠模型，该模型允许诱导和组织特异性失活 METTL3 区分 METTL3 的催化和非催化功能并了解 m6A 的作用 干细胞自我更新和分化的标志。

Project Summary RNA modifications (the epitranscriptome) represent a mechanism of post-transcriptional gene expression regulation and an emergent and exciting area of biology. N6-methyladenosine (m6A) is the most abundant post- transcriptional modification in mRNA, detected across the eukaryotic kingdom, from yeast to humans. m6A has been implicated in multiple molecular processes such as RNA splicing, RNA stability and miRNA processing and cellular functions such as meiosis, cell proliferation and embryonic stem cell differentiation, as well as disease states. The methyltransferases responsible for the m6A form a heterodimer in which METTL3 provides the catalytic activity. We and others have found that METTL3 has a positive impact in proliferation, differentiation and cell survival. Thus, we postulate that METTL3 represents a regulatory hub controlled by extra- and intracellular signals that allows the cells to respond to developmental cues and specific metabolic contexts by modulating RNA metabolism. Despite the rapid increase in our knowledge about the functions of m6A, there are many fundamental aspects of this process that remain unknown. For example, it is yet to be determined if the activity of the methyltransferases is regulated by upstream signaling pathways, and if this is the case, what are the mechanisms involved, and what are the molecular and cellular consequences of this regulation in homeostasis and development? Another key issue is to understand how the specificity of the methylation reaction is determined. We know that the recognition sequence for m6A methylation consists of a very short sequence motif – but despite the abundance of this sequence in the transcriptome only a small fraction of such motifs gets methylated. Additional RNA structural motifs or sequence requirements have not been identified. To answer these broad questions, our lab is undertaking a multidisciplinary approach that includes complementary projects in the areas of molecular and cell biology, biochemistry as well as structural studies complemented with novel engineered mouse models. The program described here will allow us to solve the mechanisms involved in the regulation of the m6A methylation pathway upon extracellular stimulation, metabolic stress and development. Using state-of-the-art proteomic techniques, we will identify context-specific post- translational modifications acquired by METTL3, and their impact on the activity and specificity of the enzyme. We will complement these studies with structural techniques including X-ray crystallography and cryo-electron microscopy to define the molecular consequences of regulatory events on protein complex formation and substrate recognition. In parallel, we will use our recently developed mouse model that allows the inducible and tissue specific inactivation of METTL3 to distinguish between catalytic and non-catalytic functions of METTL3 and to understand the role of the m6A mark in stem cell self-renewal and differentiation.