Functional alterations of the dihydrouridine landscape in response to environmental stress

二氢尿苷景观响应环境压力的功能改变

• 批准号: 10256617
• 负责人: Wendy Victoria Gilbert
• 金额: $ 25.03万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10256617
• 关键词: Affect; Alkylating Agents; Biology; Cells; Chemicals; Codon Nucleotides; Complex; Computing Methodologies; DNA; DNA Damage; Data; Development; Environmental Exposure; Enzymes; Exposure to; Future; Gene Expression; Gene Expression Regulation; Gene-Modified; Genetic Translation; Genomics; Goals; Grant; Growth; Half-Life; Health; Human; Hydrogen Peroxide; Knock-out; Knowledge; Laboratories; Link; Location; Maps; Mass Spectrum Analysis; Messenger RNA; Methods; Methyl Methanesulfonate; Modification; Molecular Conformation; Mutation; Nucleosides; Nucleotides; Output; Oxidative Stress; Physiological; Post-Transcriptional Regulation; Proteins; Publishing; RNA; RNA Splicing; RNA-Directed DNA Polymerase; RNA-Protein Interaction; Reactive Oxygen Species; Regulation; Reporting; Resistance; Resolution; Site; Site-Directed Mutagenesis; Stress; System; Technology; Toxic Environmental Substances; Toxicant exposure; Transfer RNA; Translations; Untranslated RNA; Vertebral column; Work; Yeasts; base; cell type; environmental stressor; epitranscriptome; experience; genome-wide; mRNA Stability; new technology; novel; polyadenylated messenger RNA; response; ribosome profiling; stoichiometry; toxicant; transcriptome; transcriptomics

PROJECT SUMMARY Significance: Environmental stresses that promote increases in cellular reactive oxygen species (ROS) and DNA damage reprogram certain RNA modifications and regulate gene expression. However, we currently lack knowledge of the locations and stoichiometry of many RNA modifications. This is primarily due to the lack of high-throughput methods to detect the majority of modified nucleosides. Our work seeks to map dihydrouridine (D), an intriguing and understudied RNA modification that is likely to be prevalent and regulated in mRNA as well as tRNA. We will then use systematic approaches to relate exposure-induced changes in D modifications to altered mRNA translation and stability. This work will break new ground in exposure biology and epitranscriptome studies by uncovering toxicant-induced changes in RNA modifications that alter gene expression. Approach: The goal of this exploratory project is to discover the physiologically relevant targets of dihydrouridine synthases (DUS) that show altered subcellular localization and RNA target modification following environmental exposures to toxicants that promote increased ROS or DNA damage. Loss of Dihydrouridine Synthase 3 (DUS3) leads to increased sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) in yeast whereas loss of Dihydrouridine Synthase 1 (DUS1) causes increased resistance to hydrogen peroxide (H2O2), which increases ROS and causes oxidative stress. Notably, Dus1 and Dus3/DUS3L associate with polyadenylated mRNA in yeast and various human cell types and so the D landscape is likely to be complex and include sites in mRNA that are currently undiscovered. We hypothesize that environmental stress leads to adaptive as well as pathophysiological changes in the sites and/or levels of specific dihydrouridine modifications. Aim 1 deploys new technology developed in our laboratory for comprehensive genomic analysis of dihydrouridine (D) in cells exposed to H2O2 and MMS. Aim 2 leverages this knowledge, together with systems-level analysis of mRNA translation and stability, to determine how changes in the D landscape control gene expression. Our approach exploits unique chemical features of dihydrouridine to derivatize D nucleotides, enrich for D containing RNA, and determine the locations of D with single-nucleotide resolution. Preliminary data establish selectivity for D and the ability to generate precise modification-dependent blocks to reverse transcriptase, which we will analyze by Illumina sequencing. We have assembled an outstanding team to achieve our objectives. Our laboratory is a technological pioneer in the discovery of RNA modification sites by developing experimental and computational methods to map the locations of novel mRNA modifications on a transcriptome-wide scale with single-nucleotide resolution. We are also very experienced in systems-level analysis of cellular translation and we are collaborating with an expert in mRNA stability profiling. Together, this work will reveal the changing dihydrouridine landscape in cells exposed to environmental toxicants and illuminate the underlying basis for the adaptive as well as pathophysiological effects of altered dihydrouridine synthase activity.

项目摘要 意义：促进细胞活性氧（ROS）和 DNA损伤重编程某些RNA修饰并调节基因表达。但是，我们目前缺乏 对许多RNA修饰的位置和化学计量学的了解。这主要是由于缺乏 检测大多数修饰核苷的高通量方法。我们的工作旨在映射二氢 （d），一种有趣且研究的RNA修饰，也很可能在mRNA中受到调节 作为tRNA。然后，我们将使用系统的方法将暴露诱导的D修改变化与 mRNA翻译和稳定性改变。这项工作将破坏曝光生物学和表面翻译组的新基础 通过发现毒性引起的改变基因表达的RNA修饰的变化的研究。 方法：这个探索性项目的目标是发现二氢胺的生理相关靶标 在环境之后显示出细胞下定位和RNA靶标改变的合成酶（DU） 暴露于促进ROS或DNA损伤增加的毒物。二氢苷合酶3的损失（DUS3） 导致对酵母中DNA烷基化剂甲基磺酸盐（MMS）的敏感性提高，而损失 二氢丙啶合酶1（DUS1）的含量增加了对过氧化氢的抗性（H2O2），这增加了 ROS并引起氧化应激。值得注意的是，dus1和dus3/dus3l与聚腺苷酸化mRNA相关 酵母和各种人类细胞类型，因此D景观可能很复杂，并且包括mRNA中的位置 目前未被发现。我们假设环境压力会导致自适应和 特定二氢修饰的位点和/或水平的病理生理变化。 AIM 1部署新的 在我们的实验室开发的技术，用于细胞中二氢氨基氨酸（D）的全面基因组分析 暴露于H2O2和MMS。 AIM 2利用这些知识以及系统级别的mRNA分析 翻译和稳定性，以确定D景观控制基因表达的变化。我们的方法 利用二氢胺的独特化学特征来衍生D核苷酸，富含D含有RNA的D核苷酸，并且 用单核苷酸分辨率确定D的位置。初步数据确定了D和D的选择性 能够生成精确的修饰依赖性块与逆转录酶的能力，我们将通过该酶进行分析 Illumina测序。我们组建了一支杰出的团队来实现我们的目标。我们的实验室是 通过开发实验和计算，发现RNA修饰站点的技术先驱 用单核苷酸在整个转录组范围内绘制新的mRNA修饰位置的方法 解决。我们在系统级别的细胞翻译分析中也非常有经验，我们正在协作 具有mRNA稳定性分析的专家。这项工作将揭示不断变化的二氢岛景观 在暴露于环境有毒物质的细胞中，并阐明了适应性和适应性的基础 二氢苷合酶活性改变的病理生理作用。