Molecular tools to understand cellular mRNA demethylation

项目总结/摘要 mRNA上的甲基修饰对于哺乳动物细胞的命运决定是必不可少的，并且最近已经显示 在许多人类癌症的发展中起着重要作用。去甲基酶FTO， 丰富的N6-甲基腺苷修饰，以动态调节mRNA甲基化，与启动 和癌症的进展，包括脑癌、乳腺癌、胃癌、宫颈癌、间皮瘤和白血病。最近的工作 已经表明FTO的抑制抑制胶质母细胞瘤的肿瘤进展，胶质母细胞瘤是最常见和最致命的肿瘤， 这表明FTO和其他mRNA修饰酶可能是有前途的抗癌药物 目标的然而，我们缺乏对FTO如何靶向其修饰的mRNA底物的机制理解， 影响mRNA功能，并促进疾病进展。我们的长期目标是定义生物化学， 结构和分子机制，调节mRNA去甲基化的细胞，并开发新的 描述整个转录组和不同基因组中去甲基化活性所需的工具和理解， 人类疾病状态。本建议中概述的工作将：（1）确定 使用X射线晶体学和酶学在原子水平上对FTO靶向选择性进行合成改性， 核苷酸类似物，（2）产生共价捕获FTO安装的去甲基化的生物正交化学探针 中间体，以直接定位mRNA上的去甲基化位点，以及（3）探索分子机制 通过FTO安装的去甲基化中间体可以直接调节mRNA功能。这项工作是 具有影响力和重要意义，因为它将促进该领域对甲基橡皮擦FTO如何靶向的理解 不同的mRNA修饰，以及如何未探索的，亚稳定的中间修饰安装期间， FTO介导的去甲基化影响mRNA功能。此外，我们开发的捕获去甲基化的工具 中间体对于在不同细胞类型的转录组中绘制mRNA去甲基化位点是至关重要的 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 与FTO功能有关的人类癌症。

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.