The Molecular and Regulatory Mechanism of m6A mRNA Modification

m6A mRNA修饰的分子及调控机制

• 批准号: 10188565
• 负责人: Shengdong Ke
• 金额: $ 42.5万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-12-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188565
• 关键词: Address; Base Sequence; Biochemical; Biochemistry; Biological Process; Biology; CRISPR interference; Computational Biology; Computer Models; Cytoplasm; Data; Deposition; Event; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Genomics; Goals; Half-Life; Interdisciplinary Study; Knowledge; Link; Maps; Messenger RNA; Methods; Modification; Molecular; Mutation; Nuclear Accidents; Positioning Attribute; Public Health; RNA; Role; Site; Site-Directed Mutagenesis; Work; design; genetic regulatory protein; genome-wide; high throughput screening; human disease; innovation; insight; mRNA Precursor; mRNA Stability; malignant neurologic neoplasms; nervous system disorder; novel; programs; protein complex; screening; stem cell differentiation; targeted treatment; transcription factor

PROJECT SUMMARY/ABSTRACT RNA modifications influence RNA expression and function without changing the underlying nucleotide sequence. N6-methyladenosine (m6A) is the most abundant mRNA internal modification, existing in thousands of mRNAs, of which the majority encode for regulatory proteins such as transcription factors. Although emerging evidence links m6A to stem cell differentiation, neurological disease and cancer, the molecular and regulatory mechanisms of m6A are poorly understood. To begin to address this significant knowledge gap, I previously developed the m6A-CLIP method to precisely map m6A modifications in mRNAs genome-wide, enabling the direct examination of m6A function in a site-specific manner. We discovered that m6A addition is part of mammalian pre-mRNA synthesis. We also showed that site-specific mutation of m6A sites leads to increased mRNA half-life in the cytoplasm. Thus, my work in the last five years has established a completely new role for m6A as an important link between nuclear events that occur on nascent pre-mRNA and cytoplasmic events that determine mRNA stability. These findings provide the premise for the current proposal and they uniquely position my interdisciplinary research program to carry out the proposed studies. Here, our objectives are to identify the molecular mechanisms regulating m6A site-specific deposition and m6A function in cytoplasmic mRNA stability. We will reach these objectives by biochemical characterization of the m6A protein complexes. We will also perform genome-wide CRISPR interference screening to systematically identify m6A regulators. Finally, we will build a data-driven computational model to predict m6A site-specific deposition and its effects in cytoplasmic mRNA turnover. Thus, successful completion of the proposed work will systematically reveal the underlying mechanisms of m6A RNA biology by combining cutting edge computational biology, innovative biochemistry and high-throughput genetics. These findings are expected to have a transformative impact on the RNA field by stimulating further studies into the role of mRNA modifications in regulating gene expression. Our long-term goal is to leverage the mechanistic information learned herein to gain deep insights into the role of m6A in human disease and to design novel mRNA modification-targeted therapeutics that regulate gene expression without altering the host genomic sequence.

项目总结/摘要 RNA修饰影响RNA表达和功能，而不改变潜在的核苷酸 顺序N6-甲基腺苷（m6 A）是最丰富的mRNA内部修饰，存在于数千个 其中大部分编码调节蛋白，如转录因子。虽然 新出现的证据将m6 A与干细胞分化、神经系统疾病和癌症联系起来， 对m6 A的调节机制知之甚少。为了开始解决这一重大的知识差距，我 先前开发了m6 A-CLIP方法来精确地定位mRNA全基因组中的m6 A修饰， 使得能够以位点特异性方式直接检查m6 A功能。我们发现m6 A加成是 哺乳动物前mRNA合成的一部分。我们还发现，m6 A位点的位点特异性突变导致 增加mRNA在细胞质中的半衰期。因此，我在过去五年的工作已经建立了一个完全 m6 A作为发生在新生前mRNA上的核事件和 决定mRNA稳定性的细胞质事件。这些发现为目前的提议提供了前提 它们使我的跨学科研究计划能够进行所提出的研究。这里我们的 目的是确定调节m6 A位点特异性沉积和m6 A功能的分子机制， 细胞质mRNA稳定性。我们将通过对m6 A蛋白的生物化学表征来达到这些目的 配合物我们还将进行全基因组CRISPR干扰筛选，以系统地识别m6 A 监管部门最后，我们将建立一个数据驱动的计算模型来预测m6 A特定地点的沉积， 其在细胞质mRNA周转中的作用。因此，顺利完成拟议的工作将系统地 通过结合尖端计算生物学，揭示m6 A RNA生物学的潜在机制， 创新生物化学和高通量遗传学。这些发现预计将产生变革性的影响 通过刺激对mRNA修饰在调节基因中的作用的进一步研究， 表情我们的长期目标是利用在此学到的机械信息来获得深刻的见解 研究m6 A在人类疾病中的作用，并设计新的mRNA修饰靶向治疗方法， 调节基因表达而不改变宿主基因组序列。