The molecular grammar of human RNA biology

人类RNA生物学的分子语法

• 批准号: 10622907
• 负责人: Stephen Nicholas Floor
• 金额: $ 36.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10622907
• 关键词: Address; Area; Binding; Biological Assay; Biology; Cells; Cellular biology; Couples; Deaminase; Deamination; Development; Elements; Gene Expression; Genetic; Goals; Heterogeneity; Human; Individual; Measurement; Measures; Mediating; Messenger RNA; Molecular; Molecular Biology; Proteins; RNA; RNA Sequences; RNA Stability; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Research; Ribonucleoproteins; Ribosomes; Site; Systems Biology; Therapeutic; Training; Translations; Variant; Work; human disease; method development; novel strategies; posttranscriptional; single molecule

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.

项目摘要/摘要 我的研究的总体目标是定义RNA分子的分子语法如何调节 基因表达。为了实现这一目标，我在 分子，细胞和系统生物学对人RNA生物学的理解。我试图建立在我的基础上 RNA生物学和方法开发中的记录记录，以解决RNA中的紧迫问题 生物学。该提案确定了未来五年对查询的两个强调领域。第一个 该提案的重点是我通过识别的十年研究RNA结合蛋白DDX3的研究 并探索理解中的关键差距。 DDX3是必不可少的ATP依赖性RNA结合蛋白 夫妻ATP与局部RNA双链体结合和核糖核蛋白重塑。我的事先工作 小组和其他人已经实施了DDX3，以用于转化控制的mRNA分子，其中包含多种 mRNA元素，但是精确的机制，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，我们试图定义DDX3如何与核糖体相互作用以介导 翻译控制，使用我们开发的新测定法以无偏见定义DDX3依赖性翻译 方式，定义DDX3与遗传相互作用不同的错义变体的耗竭以及 建立导致DDX3耗竭后RNA水平变化的机制。第二强 该提案在我的小组中推进了新的发展，该发展能够对RNA-的单分子测量 细胞中的蛋白质相互作用。我们进化了一个新的基于死亡的分子记录器以捕获RNA-蛋白 通过修改与RNA蛋白相互作用相邻的RNA的序列来相互作用。通过长阅读 然后，我们可以识别由用脱氨酶标记的蛋白质结合的RNA中的区域。 使用这种方法，我们发现在单个mRNA上的RNA结合蛋白位点中的意外异质性 分子。我们建议以这些发现为基础，以了解机械和功能 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结合的机制，提出的研究与我的研究目标一致 蛋白质并通过开发新方法来测量单分子RNA生物学。我希望结果 提出的研究旨在提高对RNA生物学的理解，对基本的影响 了解RNA，人类疾病和mRNA疗法。