Molecular tools to understand cellular mRNA demethylation

了解细胞 mRNA 去甲基化的分子工具

• 批准号: 10026274
• 负责人: Jeffrey Scott Mugridge
• 金额: $ 24.07万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10026274
• 关键词: Acute Myelocytic Leukemia; Antineoplastic Agents; Base Pairing; Binding; Biochemical; Biological; Brain; Breast; Cell Proliferation; Cell Survival; Cells; Cervical; Chemicals; Collaborations; Data; Disease; Disease Progression; Drug Targeting; Enzymatic Biochemistry; Enzymes; Event; Fatty acid glycerol esters; Glioblastoma; Goals; Human; Link; Malignant Neoplasms; Malignant neoplasm of brain; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Mesothelioma; Messenger RNA; Methylation; Modification; Molecular; Molecular Probes; Mus; Mutation; Obesity; Phase; Play; Proteins; RNA; RNA Caps; RNA methylation; RNA-Binding Proteins; Reader; Regulation; Role; Shapes; Site; Stomach; Structure; Surface; Testing; Therapeutic; Thermodynamics; Transcript; Transfer RNA; Universities; Work; X-Ray Crystallography; analog; base; cell growth; cell type; demethylation; embryonic stem cell; experimental study; human disease; leukemia; mRNA capping; malignant breast neoplasm; novel strategies; nucleotide analog; stem cell differentiation; stem cells; structured data; therapeutic target; tool; transcriptome; transcriptome sequencing; tumor progression

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.

项目摘要 /摘要 mRNA上的甲基修饰对于哺乳动物细胞脂肪决策至关重要，最近已显示 在许多人类癌症的发展中扮演重要角色。脱甲基酶FTO擦除 丰富的N6-甲基二腺苷对动态调节mRNA甲基化的修饰与起始有关 以及包括大脑，乳房，胃，宫颈，间皮瘤和白血病等癌症的进展。最近的工作 已经表明，FTO抑制抑制胶质母细胞瘤的肿瘤进展，最常见和最致命 脑癌的形式，表明FTO和其他mRNA修改酶可能是有希望的抗癌药物 目标。但是，我们对FTO如何靶向其修饰的mRNA底物，缺乏机械理解， 影响mRNA功能，并导致疾病进展。我们的长期目标是定义生化， 调节细胞中mRNA脱甲基化并发展新的结构和分子机制 在整个转录组和潜水员中表征脱甲基化活动所需的工具和理解 人类疾病状态。该提案中概述的工作将：（1）定义 FTO使用X射线晶体学和合成修饰的酶学在原子水平上进行选择性 核苷酸类似物，（2）产生生物正交化学问题，共价捕获FTO安装的脱甲基化 中间体直接在mRNA上绘制脱甲基化位点，（3）探索分子机制 FTO安装的脱甲基化中间体可以直接调节mRNA功能。这项工作是 有影响力和意义，因为它将提高该领域对甲基橡皮橡胶FTO的理解 进行不同的mRNA修饰以及在 FTO介导的脱甲基影响mRNA功能。此外，我们开发的工具以捕获脱甲基化 中间体对于在不同细胞类型的转录组中绘制mRNA去甲基化的位点至关重要 和疾病状态，这将使我们的实验室和其他人可以直接评估mRNA脱甲基的作用 与FTO功能相关的人类癌症。