# Functional alterations of the dihydrouridine landscape in response to environmental stress

> **NIH NIH R21** · YALE UNIVERSITY · 2020 · $243,369

## Abstract

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. Togethe...

## Key facts

- **NIH application ID:** 9930352
- **Project number:** 1R21ES031525-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Wendy Victoria Gilbert
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $243,369
- **Award type:** 1
- **Project period:** 2020-09-08 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9930352

## Citation

> US National Institutes of Health, RePORTER application 9930352, Functional alterations of the dihydrouridine landscape in response to environmental stress (1R21ES031525-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9930352. Licensed CC0.

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