Mechanisms of Regulation of the m6A mRNA Methylation Machinery

项目摘要 RNA修饰（表转录组）代表了转录后基因表达的机制 调节和一个新兴的令人兴奋的生物学领域。N6-甲基腺苷（m6 A）是最丰富的后- mRNA的转录修饰，在真核生物界检测到，从酵母到人类。m6 a是 参与多个分子过程，如RNA剪接、RNA稳定性和miRNA加工，以及细胞 功能，如减数分裂，细胞增殖和胚胎干细胞分化，以及疾病状态。的 负责m6 A的甲基转移酶形成异二聚体，其中胃L3提供催化活性。我们 和其他人已经发现，胃L3在增殖、分化和细胞存活中具有积极影响。因此我们 假设胃L3代表了一个由细胞外和细胞内信号控制的调节中心， 这些细胞通过调节RNA代谢来响应发育线索和特定的代谢环境。 尽管我们对m6 A功能的了解迅速增加，但这一过程仍有许多基本方面。 过程仍然未知。例如，还有待确定甲基转移酶的活性是否是 受上游信号通路调节，如果是这种情况，涉及的机制是什么， 这种调节在体内平衡和发育中的分子和细胞后果？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， m6 A甲基化由一个非常短的序列基序组成，但尽管在哺乳动物中该序列丰富， 在转录组中，只有一小部分这样的基序被甲基化。其他RNA结构基序或序列 需求尚未确定。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研究。 方法，包括分子和细胞生物学，生物化学以及 结构研究补充了新的工程小鼠模型。这里描述的程序将允许我们 解决了在细胞外刺激时调节m6 A甲基化途径所涉及的机制， 代谢应激和发育。使用最先进的蛋白质组学技术，我们将确定特定的上下文后， 通过胃L3获得的翻译修饰，及其对酶活性和特异性的影响。我们将 补充这些研究与结构技术，包括X射线晶体学和冷冻电子显微镜， 定义蛋白质复合物形成和底物识别的调控事件的分子后果。在 与此同时，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 区分胃L3的催化和非催化功能，并了解m6 A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.