The Molecular and Regulatory Mechanism of m6A mRNA Modification

项目摘要/摘要 RNA修饰影响RNA的表达和功能，而不改变潜在的核苷酸 序列。N6-甲基腺苷(M6A)是一种含量最丰富的mRNA内部修饰，存在于数千种DNA中 MRNAs，其中大部分编码转录因子等调节蛋白。虽然 新出现的证据表明，m6A与干细胞分化、神经疾病和癌症有关，分子和 对m6A的调控机制知之甚少。为了开始解决这一重大的知识差距，我 先前开发了m6A-CLIP方法以精确地定位全基因组mRNAs中的m6A修饰， 能够以特定于现场的方式直接检查M6A功能。我们发现m6A的添加是 哺乳动物前信使核糖核酸合成的一部分。我们还发现，m6A位点的定点突变会导致 增加了细胞质中的mRNA半衰期。因此，我在过去五年的工作已经完全建立了 M6A作为发生在新生前-mRNA上的核事件和核事件之间的重要联系的新角色 决定信使核糖核酸稳定性的细胞质事件。这些发现为当前的提案提供了前提 他们将我的跨学科研究项目置于独特的位置，以实施拟议的研究。在这里，我们的 目的是确定调控M6A位点特异性沉积和M6A功能的分子机制。 细胞质mRNA的稳定性。我们将通过对m6a蛋白进行生化表征来实现这些目标。 复合体。我们还将进行全基因组CRISPR干扰筛查，以系统地鉴定m6A 监管者。最后，我们将建立一个数据驱动的计算模型来预测m6A特定地点的沉积和 其在细胞质mRNA周转中的作用。因此，拟议工作的圆满完成将有系统地 通过结合尖端计算生物学揭示m6A RNA生物学的潜在机制， 创新的生物化学和高通量遗传学。预计这些发现将产生革命性的影响 促进进一步研究信使核糖核酸修饰在基因调控中的作用对核糖核酸领域的影响 表情。我们的长期目标是利用在这里学到的机械性信息来获得深刻的洞察力 研究m6A在人类疾病中的作用，并设计新的以mRNA修饰为靶向的治疗方法 在不改变宿主基因组序列的情况下调节基因表达。

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.