# Developing Tools to Map and Quantify Dihydrouridine in the Mammalian Transcriptome

> **NIH NIH F31** · YALE UNIVERSITY · 2020 · $45,520

## Abstract

Project Summary/Abstract
Recently, several canonical tRNA modifications, including pseudouridine (Ψ ), N6-methyladenosine (m6A), N1-
methyladenosine (m1A), and 5-methylcytosine (m5C) have been found in messenger RNA. The development of
new high-throughput sequencing methods has revealed the location of individual mRNA modifications and
paved the way for the interrogation of their function across the transcriptome. RNA modifications have multiple
effects on mRNA during its lifespan: splicing, trafficking, translation and degradation can all be altered.
Dihydrouridine (D) is a ubiquitous modified nucleotide found in tRNAs in every branch of the tree of life.
Conserved dihydrouridine synthases (DUS) enzymes DUS1L and DUS3L, associate with mRNA in mammalian
cells, suggesting that they modify mRNA. Dihydrouridine affects RNA secondary structure by distorting the
pyrimidine ring, which is likely to profoundly influence mRNA metabolism. My preliminary data demonstrate
that D is installed in mRNAs in yeast, and I hypothesize that this is also true in mammalian cells. I have
developed a method (D-seq) to profile D at single-nucleotide resolution. The method works by reducing D with
sodium borohydride, causing reverse transcriptase (RT) to fall off the template while traversing reduced D. To
expand this technique to transcriptome scale, I will develop a two-step protocol to directly couple biotin to
reduced D (eD-seq). This will permit enrichment of RNAs containing D, and comprehensive inspection of all
RNA in the human transcriptome for D. To quantify the stoichiometry of modification at D positions, I will also
develop a modified form of D-Seq that uses a different RT which misincorporates while traversing D (D-MaP-
seq). To demonstrate the utility of these methods, and identify disease relevant D sites, I will comprehensively
map and quantify D in the transcriptome of two types of cancer that have characteristic overexpression of
DUS. The new D profiling tools that I will develop will identify the locations of DUS-dependent Ds across the
transcriptome. They are likely to reveal that D is a previously unknown component of the human
`epitranscriptome'.

## Key facts

- **NIH application ID:** 10066046
- **Project number:** 1F31CA254339-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Austin Stratton Draycott
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $45,520
- **Award type:** 1
- **Project period:** 2020-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10066046

## Citation

> US National Institutes of Health, RePORTER application 10066046, Developing Tools to Map and Quantify Dihydrouridine in the Mammalian Transcriptome (1F31CA254339-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10066046. Licensed CC0.

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