Molecular tools to understand cellular mRNA demethylation

项目摘要/摘要 对信使核糖核酸的甲基化修饰对哺乳动物细胞命运的决定是必不可少的，最近的研究表明 在许多人类癌症的发展过程中发挥重要作用。去甲基酶FTO，它可以擦除 丰富的N6-甲基腺苷修饰动态调节mRNA甲基化，与启动有关 以及癌症的进展，包括脑癌、乳腺癌、胃癌、宫颈癌、间皮瘤和白血病。近期工作 研究表明，抑制FTO可以抑制最常见和最致命的胶质母细胞瘤的肿瘤进展 脑癌的形式，提示FTO和其他mRNA修饰酶可能是有前途的抗癌药物 目标。然而，我们缺乏对FTO如何靶向其修饰的mRNA底物的机械理解， 影响信使核糖核酸的功能，并有助于疾病的进展。我们的长期目标是定义生物化学， 调节细胞内信使核糖核酸去甲基化的结构和分子机制，并开发新的 确定转录组和不同物种脱甲基化活性所需的工具和理解 人类疾病状态。本提案中概述的工作将：(1)确定 合成修饰的X射线结晶学和酶学在原子水平上对FTO靶向的选择性 核苷酸类似物，(2)产生共价捕获FTO安装的脱甲基化的生物正交化学探针 中间体直接定位基因去甲基化位点；(3)探讨分子机制 FTO安装的去甲基化中间体可能通过它直接调节mRNA的功能。这项工作是 影响和意义重大，因为它将促进该领域对甲基橡皮擦FTO目标的理解 到不同的信使核糖核酸修饰，以及未开发的、亚稳定的中间修饰是如何在 FTO介导的去甲基化影响了mRNA的功能。此外，我们开发的捕捉去甲基化的工具 中间体对于在不同细胞类型的转录组中定位mrna去甲基化的位置是至关重要的。 和疾病状态，这将使我们的实验室和其他人能够直接评估mRNA去甲基化在 人类癌症与FTO功能有关。

PROJECT SUMMARY / ABSTRACT Methyl modifications on mRNA are essential for mammalian cell fate decisions and have recently been shown to play important roles in the progression of many human cancers. The demethylase FTO, which erases abundant N6-methyladenosine modifications to dynamically regulate mRNA methylation, is linked to initiation and progression of cancers including brain, breast, gastric, cervical, mesothelioma, and leukemia. Recent work has shown that inhibition of FTO suppresses tumor progression in glioblastoma, the most common and deadliest form of brain cancer, suggesting FTO and other mRNA-modifying enzymes may be promising anti-cancer drug targets. However, we lack a mechanistic understanding of how FTO targets its modified mRNA substrates, impacts mRNA function, and contributes to disease progression. Our long-term goal is to define the biochemical, structural, and molecular mechanisms that regulate mRNA demethylation in the cell, and to develop the new tools and understanding needed to characterize demethylation activity across the transcriptome and in diverse human disease states. The work outlined in this proposal will: (1) define the structural and biochemical basis of FTO target selectivity at the atomic level using X-ray crystallography and enzymology with synthetic modified nucleotide analogs, (2) generate bioorthogonal chemical probes that covalently trap FTO-installed demethylation intermediates to directly map sites of demethylation on mRNA, and (3) explore the molecular mechanisms through which FTO-installed demethylation intermediates may directly regulate mRNA function. This work is impactful and significant because it will advance the field’s understanding of how methyl eraser FTO is targeted to different mRNA modifications and how unexplored, metastable intermediate modifications installed during FTO-mediated demethylation impact mRNA function. Furthermore, the tools we develop to trap demethylation intermediates will be critical to map sites of mRNA demethylation across the transcriptome in different cell types and disease states, which will allow our lab and others to directly assess the role of mRNA demethylation in human cancers linked to FTO function.