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.

项目概要 意义：环境压力会促进细胞活性氧（ROS）和 DNA 损伤重新编程某些 RNA 修饰并调节基因表达。然而我们目前缺乏 许多 RNA 修饰的位置和化学计量的知识。这主要是由于缺乏 高通量方法检测大多数修饰核苷。我们的工作旨在绘制二氢尿苷图谱 (D)，一种有趣且尚未被研究的 RNA 修饰，它也可能在 mRNA 中普遍存在并受到调节 作为 tRNA。然后，我们将使用系统方法将暴露引起的 D 修饰变化与 改变 mRNA 翻译和稳定性。这项工作将在暴露生物学和表观转录组领域开辟新天地 通过揭示毒物诱导的 RNA 修饰变化来改变基因表达的研究。 方法：该探索性项目的目标是发现二氢尿苷的生理相关靶点 合酶 (DUS) 表现出环境变化后亚细胞定位和 RNA 靶标修饰的改变 接触会促进 ROS 或 DNA 损伤增加的有毒物质。二氢尿苷合酶 3 (DUS3) 丢失 导致酵母中对 DNA 烷化剂甲磺酸甲酯 (MMS) 的敏感性增加，而损失 二氢尿苷合酶 1 (DUS1) 的减少会导致对过氧化氢 (H2O2) 的抵抗力增加，从而增加 ROS 并引起氧化应激。值得注意的是，Dus1 和 Dus3/DUS3L 与多腺苷酸化 mRNA 相关 酵母和各种人类细胞类型，因此 D 景观可能很复杂，包括 mRNA 中的位点 目前尚未发现。我们假设环境压力会导致适应性和 特定二氢尿苷修饰的位点和/或水平的病理生理学变化。目标 1 部署新 我们实验室开发的技术，用于细胞中二氢尿苷 (D) 的全面基因组分析 暴露于 H2O2 和 MMS。目标 2 利用这些知识以及 mRNA 的系统级分析 翻译和稳定性，以确定 D 景观的变化如何控制基因表达。我们的方法 利用二氢尿苷的独特化学特征衍生化 D 核苷酸，富集含有 D 的 RNA，以及 以单核苷酸分辨率确定 D 的位置。初步数据确定了 D 和 生成精确的逆转录酶修饰依赖块的能力，我们将通过以下方式进行分析 Illumina 测序。我们组建了一支优秀的团队来实现我们的目标。我们的实验室是一个 通过开发实验和计算发现 RNA 修饰位点的技术先驱 用单核苷酸在转录组范围内绘制新 mRNA 修饰位置的方法 解决。我们在细胞翻译的系统级分析方面也非常有经验，并且我们正在合作 与 mRNA 稳定性分析专家合作。这项工作将共同揭示不断变化的二氢尿苷景观 在暴露于环境毒物的细胞中，阐明了适应性和适应性的潜在基础 二氢尿苷合酶活性改变的病理生理学影响。