Selectivity and regulation of mRNA demethylation by iron-dependent dioxygenases

铁依赖性双加氧酶对 mRNA 去甲基化的选择性和调节

• 批准号: 10620782
• 负责人: Jeffrey Scott Mugridge
• 金额: $ 39.13万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10620782
• 关键词: Acute Myelocytic Leukemia; Ascorbic Acid; Biochemistry; Bioinorganic Chemistry; Biological; Biology; Cells; Cellular biology; Chemicals; Dioxygenases; Disease; Enzymes; Family; Family member; Glioblastoma; Goals; Human; Iron; Link; Malignant Neoplasms; Mammalian Cell; Maps; Messenger RNA; Modification; Molecular; Monitor; Pathway interactions; Play; RNA; RNA methylation; Regulation; Research; Role; Structure; Transcript; Visualization; cofactor; demethylation; human disease; insight; mRNA capping; novel; novel therapeutic intervention; overexpression; programs; structural biology; therapeutic target; tool; transcriptome; tumor growth; tumor progression; tumorigenesis

TITLE: Selectivity and regulation of mRNA demethylation by iron-dependent dioxygenases ABSTRACT: The long-term goals of this research program are to (1) define the structural and molecular mechanisms that control the selectivity and function of RNA demethylase enzymes, (2) develop new chemical tools to monitor RNA demethylation in cells, and (3) understand how the key cofactor ascorbic acid interacts with RNA demethylases and other iron-dependent dioxygenase family members to regulate their activity. Methyl modifications on mRNA tune transcript function, are essential for mammalian cell fate decisions, and play important roles in the progression of many human cancers. The iron-dependent dioxygenase enzymes FTO and AlkBH5 act as ‘erasers’ of highly abundant N6-methyladenosine (m6A) modifications found in the mRNA body and, in the case of FTO, N6,2’-O-dimethyladenosine (m6Am) modifications found on the 5’ mRNA cap. These RNA demethylases are overexpressed in cancers including glioblastoma and acute myeloid leukemia, where increased demethylation activity and reduced methyl modification levels promote tumorigenesis and cancer progression. Despite these clear links to human disease, we currently have a poor understanding of how FTO and AlkBH5 recognize their biological substrates, which mRNA transcripts are targeted for demethylation, and how demethylation influences mRNA function. Furthermore, FTO and AlkBH5 belong to the non-heme iron(II) and -ketoglutarate-dependent family of dioxygenases that require ascorbic acid (vitamin C) as a cofactor for efficient activity, but we have almost no structure-level insights into how ascorbic acid interacts with this diverse family of enzymes and how this physical interaction may potentiate dioxygenase activity in cells. This proposal combines approaches from biochemistry, structural biology, chemical biology, bioinorganic chemistry, and cell biology to determine the structural basis for RNA demethylase selectivity, develop novel probes to map demethylation targets across the transcriptome, and quantify and visualize the dioxygenase-ascorbic acid interaction to understand how this cofactor regulates enzymatic activity. The results from these proposed studies will significantly enhance our understanding of how cellular mRNA demethylation is regulated in cells and pave the way for therapeutics that target demethylation pathways in challenging cancers such as glioblastoma.

标题：铁依赖性双加氧酶对 mRNA 去甲基化的选择性和调节 摘要：本研究计划的长期目标是（1）定义结构和 控制 RNA 去甲基酶的选择性和功能的分子机制，(2) 开发新的化学工具来监测细胞中的 RNA 去甲基化，以及 (3) 了解 关键辅助因子抗坏血酸与 RNA 去甲基酶和其他铁依赖性酶相互作用 双加氧酶家族成员来调节其活性。 mRNA 调谐的甲基修饰 转录功能对于哺乳动物细胞的命运决定至关重要，并在 许多人类癌症的进展。铁依赖性双加氧酶 FTO 和 AlkBH5 充当高度丰富的 N6-甲基腺苷 (m6A) 修饰的“擦除器” mRNA 体以及 FTO 的 N6,2'-O-二甲基腺苷 (m6Am) 修饰发现于 5' mRNA 帽。这些 RNA 去甲基化酶在包括胶质母细胞瘤在内的癌症中过度表达 和急性髓系白血病，其中去甲基化活性增加，甲基化减少 修饰水平促进肿瘤发生和癌症进展。尽管有这些明确的链接 人类疾病，我们目前对 FTO 和 AlkBH5 如何识别它们的了解甚少 生物底物，哪些 mRNA 转录物是去甲基化的目标，以及如何去甲基化 去甲基化影响 mRNA 功能。此外，FTO和AlkBH5属于非血红素 需要抗坏血酸（维生素 C）作为高效活动的辅助因素，但我们几乎没有结构层面的见解来了解如何 抗坏血酸与这种不同的酶家族相互作用以及这种物理相互作用如何可能 增强细胞中的双加氧酶活性。该提案结合了生物化学的方法， 结构生物学、化学生物学、生物无机化学和细胞生物学，以确定 RNA 去甲基化酶选择性的结构基础，开发新型探针来绘制去甲基化图谱 跨转录组的目标，并对双加氧酶-抗坏血酸进行量化和可视化 相互作用以了解该辅助因子如何调节酶活性。这些结果 拟议的研究将显着增强我们对细胞 mRNA 如何 去甲基化在细胞中受到调节，为靶向去甲基化的治疗铺平了道路 胶质母细胞瘤等具有挑战性的癌症的途径。