Functional alterations of the dihydrouridine landscape in response to environmental stress

项目总结 意义：环境压力促进细胞内活性氧物种(ROS)的增加和 DNA损伤使某些RNA修饰重新编程，并调节基因表达。然而，我们目前缺乏 了解许多RNA修饰的位置和化学计量学。这主要是由于缺乏 高通量检测大多数修饰核苷的方法。我们的工作是绘制二氢尿苷图谱 (D)，一种耐人寻味且研究不足的RNA修饰，很可能在mRNA中普遍存在并受到调控 作为tRNA。然后，我们将使用系统的方法将暴露引起的D修饰的变化与 改变了信使核糖核酸的翻译和稳定性。这项工作将在暴露生物学和表位编码组方面开辟新的天地 通过揭示毒物引起的改变基因表达的RNA修饰的变化进行的研究。 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。 环境后亚细胞定位和RNA靶标修饰改变的合成酶(DU) 暴露在促进ROS或DNA损伤增加的毒物中。二氢尿苷合成酶3(DUS3)缺失 导致酵母对DNA烷化剂甲基甲烷磺酸(MMS)的敏感性增加，而损失 二氢尿苷合成酶1(DUS1)的表达增加了对过氧化氢(H2O2)的抵抗力，从而增加了 ROS并导致氧化应激。值得注意的是，Dus1和Dus3/DUS3L与多腺化的mRNA在 酵母和各种人类细胞类型，因此D图谱可能是复杂的，包括mRNA中的位点 目前还未被发现的生物。我们假设环境压力会导致适应性以及 二氢尿苷修饰部位和/或水平的病理生理变化。AIM 1部署新的 本实验室开发的细胞内二氢尿苷(D)全基因组分析技术 暴露在过氧化氢和MMS中。AIM 2利用了这一知识，以及对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的表达。我们的方法 利用二氢尿苷的独特化学特征来衍生D核苷酸，富含D的RNA，以及 用单核苷酸分辨确定D的位置。初步数据确定了D和 能够生成精确的依赖于修饰的块来逆转录酶，我们将通过 Illumina测序。我们已经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技术发现RNA修饰位点的技术先驱 方法用单核苷酸在转录组范围内定位新的mRNA修饰的位置 决议。我们在细胞翻译的系统级分析方面也非常有经验，我们正在合作 有一位信使核糖核酸稳定性分析专家。总之，这项工作将揭示二氢尿苷景观的变化。 在暴露于环境毒物的细胞中，并阐明了适应性以及 二氢尿苷合成酶活性改变的病理生理效应。

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.