The molecular grammar of human RNA biology

项目概要/摘要 我研究的首要目标是确定RNA分子的分子语法如何调节 基因表达。为了实现这一目标，我接受了广泛的跨学科培训， 分子、细胞和系统生物学对人类RNA生物学的理解。我寻求建立在我的 在RNA生物学和方法开发方面有记录的跟踪记录，以解决RNA中的紧迫问题 生物学这项建议确定了今后五年调查的两个重要重点领域。第一 这个建议的重点建立在我对RNA结合蛋白DDX 3的十年研究的基础上， and exploring探索critical关键gaps差距in understanding理解. DDX 3是一种必需的ATP依赖性RNA结合蛋白， 偶联ATP结合到局部RNA双链体解旋和核糖核蛋白重塑。我以前的工作 研究小组和其他人已经暗示DDX 3参与了含有多种转录因子的mRNA分子的翻译控制。 mRNA元件，但精确的机制，重要的mRNA特征和遗传相互作用仍然存在 不完全理解。在这里，我们试图确定DDX 3如何与核糖体相互作用，以介导 翻译控制，使用我们开发的一种新的检测方法来定义DDX 3依赖性翻译， 方式，以确定DDX 3与错义变体的消耗如何在遗传相互作用中不同，以及 建立导致DDX 3耗尽后RNA水平变化的机制。第二个重大问题 这个建议在我的小组中推进了新的发展，使RNA的单分子测量成为可能， 细胞中的蛋白质相互作用。我们开发了一种新的基于脱氨基的分子记录器来捕获RNA-蛋白质 通过修饰与RNA-蛋白质相互作用相邻的RNA的序列来调节RNA-蛋白质相互作用。通过长期阅读 通过测序，我们可以识别出RNA中被脱氨酶标记的蛋白质结合的区域。 使用这种方法，我们发现了个体mRNA上RNA结合蛋白位点的意外异质性， 分子。我们建议在这些发现的基础上，既要了解机制和功能， 这种异质性的影响，并将我们的方法扩展到新的RNA结合蛋白。总体看 通过定义重要的RNA结合机制，我提出的研究与我的研究目标一致 蛋白质和开发新的方法来测量单分子RNA生物学。我期待着 拟议的研究，以促进对RNA生物学的理解与影响的基本 理解RNA、人类疾病和mRNA治疗。

Project Summary/Abstract The overarching goal of my research is to define how the molecular grammar of RNA molecules regulates gene expression. To accomplish this goal, I have pursued extensive and interdisciplinary training in the molecular, cellular, and systems biology understanding of human RNA biology. I seek to build upon my documented track record in RNA biology and method development to address pressing questions in RNA biology. This proposal identifies two emphasis areas of importance for inquiry in the next five years. The first emphasis of this proposal builds on my decade of research into the RNA–binding protein DDX3 by identifying and exploring critical gaps in understanding. DDX3 is an essential ATP-dependent RNA–binding protein that couples ATP binding to local RNA duplex unwinding and ribonucleoprotein remodeling. Prior work from my group and others has implicated DDX3 in translational control for mRNA molecules containing a variety of mRNA elements, but the precise mechanism, important mRNA features, and genetic interactions remain incompletely understood. Here, we seek to define how DDX3 interacts with the ribosome to mediate translational control, to use a new assay we developed to define DDX3-dependent translation in an unbiased manner, to define how depletion of DDX3 versus missense variants differ in genetic interactions, and to establish the mechanism leading to changes in RNA levels following DDX3 depletion. The second emphasis of this proposal advances new developments in my group that enable single-molecule measurement of RNA- protein interactions in cells. We evolved a new deamination-based molecular recorder to capture RNA-protein interactions by modifying the sequence of RNA adjacent to an RNA-protein interaction. Through long-read sequencing we can then identify regions in RNA that were bound by a protein tagged with the deaminase. Using this approach, we find unexpected heterogeneity in RNA–binding protein sites on individual mRNA molecules. We propose to build upon these findings, both to understand the mechanistic and functional implications of this heterogeneity and to extend our approach to new RNA–binding proteins. Overall, the proposed research is aligned with my research goal by defining the mechanism of important RNA–binding proteins and by developing new approaches to measure single-molecule RNA biology. I expect the results of the proposed research to advance the understanding of RNA biology with implications for the fundamental understanding of RNA, human disease, and mRNA therapeutics.