The molecular grammar of human RNA biology

项目摘要/摘要 我研究的首要目标是定义RNA分子的分子语法是如何调节的 基因表达。为了实现这一目标，我进行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。我寻求在我的基础上 记录了RNA生物学和方法开发方面的记录，以解决RNA中的紧迫问题 生物学。该提案确定了今后五年调查的两个重要重点领域。第一 这项建议的重点建立在我十年来对RNA结合蛋白DDX3的研究基础上，通过识别 以及探索理解中的关键差距。DDX3是一种重要的依赖于ATP的RNA结合蛋白 将ATP结合到局部RNA双链解离和核糖核蛋白重塑。我之前的工作 小组和其他人已经将DDX3牵连到对包含各种不同的 信使核糖核酸元件，但其确切的机制、重要的信使核糖核酸特性和遗传交互作用仍然存在 不完全理解。在这里，我们试图定义DDX3如何与核糖体相互作用来调节 翻译对照，使用我们开发的一种新的分析方法来定义无偏见的DDX3依赖的翻译 方式，以定义DDX3耗尽与错义变体在遗传相互作用中的差异，并 建立导致DDX3耗尽后RNA水平变化的机制。第二个重点是 这项提议推动了我的团队的新发展，使单分子RNA测量成为可能-- 细胞内的蛋白质相互作用。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 通过修改与RNA-蛋白质相互作用相邻的RNA序列来实现相互作用。通过长期阅读 测序后，我们就可以确定RNA中与脱氨酶标记的蛋白质结合的区域。 使用这种方法，我们发现单个mrna上的rna结合蛋白位置意外地异质性。 分子。我们建议在这些发现的基础上，既理解机械的，也理解功能的 这种异质性的含义，并将我们的方法扩展到新的RNA结合蛋白。总体而言， 通过定义重要的RNA结合机制，提出的研究与我的研究目标是一致的 并开发了测量单分子RNA生物学的新方法。我期待的结果是 拟议中的研究旨在促进对RNA生物学的理解，并对基础 了解核糖核酸、人类疾病和信使核糖核酸疗法。

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.