Functional alterations of the dihydrouridine landscape in response to environmental stress

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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.