Mechanisms of Regulation of the m6A mRNA Methylation Machinery

项目摘要 RNA修饰(表位转录组)代表了转录后基因表达的一种机制 监管和生物学的一个新兴和令人兴奋的领域。N6-甲基腺苷(M6A)是体内含量最丰富的后遗症。 在真核生物王国，从酵母到人类，都能检测到mRNA的转录修饰。M6A已经成为 与多种分子过程有关，如RNA剪接、RNA稳定性、miRNA加工和细胞 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。这个 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 还有一些人发现，METTL3对细胞的增殖、分化和存活有积极的影响。因此，我们 假设METTL3代表由细胞外和细胞内信号控制的调节中枢，从而允许 细胞通过调节RNA代谢对发育线索和特定的代谢环境做出反应。 尽管我们对m6A功能的了解迅速增加，但这其中有许多基本的方面 仍然未知的过程。例如，甲基转移酶的活性是否为 由上游信号通路调节，如果是这样，涉及的机制是什么，又是什么 这种调节在动态平衡和发育中的分子和细胞后果是什么？另一个关键问题是 了解甲基化反应的特异性是如何确定的。我们知道识别序列 M6A甲基化由一个非常短的序列基序组成-但尽管该序列在 转录组只有一小部分这样的基序被甲基化。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，我们的实验室正在进行一项多学科的 包括在分子和细胞生物学、生物化学以及 结构研究与新的工程小鼠模型相辅相成。这里描述的程序将使我们能够 解决m6A甲基化途径对细胞外刺激的调节机制， 代谢应激与发育。使用最先进的蛋白质组学技术，我们将识别特定于上下文的后 METTL3获得的翻译修饰及其对酶活性和特异性的影响。我们会 用X射线结晶学和低温电子显微镜等结构技术补充这些研究 确定调控事件对蛋白质复合体形成和底物识别的分子后果。在……里面 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灭活 以区分METTL3的催化和非催化功能，并了解M6A的作用 标记干细胞的自我更新和分化。

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.