Selectivity and regulation of mRNA demethylation by iron-dependent dioxygenases

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）确定结构和 控制RNA去甲基化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 关键辅因子抗坏血酸与RNA脱甲基酶和其他铁依赖性酶相互作用 双加氧酶家族成员调节其活性。mRNA调变的甲基修饰 转录功能，是哺乳动物细胞命运决定所必需的，并在以下方面发挥重要作用： 许多人类癌症的进展。铁依赖性双加氧酶FTO和 AlkBH 5充当在细胞中发现的高度丰富的N6-甲基腺苷（m6 A）修饰的“擦除器”。 mRNA体，以及在FTO的情况下，在mRNA体上发现的N6，2 '-O-二甲基腺苷（m6 Am）修饰， 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表达 和急性髓性白血病，其中去甲基化活性增加，甲基化活性减少， 修饰水平促进肿瘤发生和癌症进展。尽管有这些明显的联系， 尽管FTO和AlkBH 5是人类疾病的一部分，但我们目前对FTO和AlkBH 5如何识别它们的 生物底物，哪些mRNA转录本是去甲基化的靶向，以及如何去甲基化， 去甲基化影响mRNA功能。此外，FTO和AlkBH 5属于非血红素 铁（II）和β-酮戊二酸依赖性双加氧酶家族需要抗坏血酸（维生素 C）作为有效活动的辅助因子，但我们几乎没有结构层面的见解， 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 增强细胞中的双加氧酶活性。这个建议结合了生物化学， 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，开发新的探针来定位去甲基化 目标在转录组，并量化和可视化双加氧酶-抗坏血酸 相互作用，以了解这种辅因子如何调节酶活性。结果从这些 这项研究将大大增强我们对细胞mRNA 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。 在具有挑战性的癌症如胶质母细胞瘤中的途径。

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.