Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA

Abstract
Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

Introduction
Dihydrouridine (D) is a modified version of uridine that is installed by dihydrouridine synthase (DUS) enzymes in all domains of life. It is of great interest to determine the locations of D modifications because elevated expression of DUS and elevated D levels in tumors are associated with worse outcomes for patients in lung [1], liver [2], and kidney [3,4] cancer. DUS target sites in tRNAs are best characterized in budding yeast [5,6] and include multiple positions within the eponymous D loop as well as sites in the variable loops of some tRNAs. D has also been detected in the genomic RNA of Dengue, Zika, Hepatitis C, and Polio viruses [7], but the specific locations are unknown. It is likely that DUS modify additional classes of cellular RNA as recently discovered for other tRNA-modifying enzymes [8]. Notably, DUS1 and DUS3 cross-link to mRNA in both yeast and human cells [9,10] suggesting their potential to modify mRNA target sites.

Results and discussion
In light of previous work showing that DUS1 and DUS3 cross-link to mRNA in both yeast and human cells [9,10], we performed bulk nucleotide analysis on RNA from budding yeast. We purified polyA+ mRNA from a dus1Δ dus2Δ dus3Δ dus4Δ quadruple mutant strain lacking all DUS activity [6] and a matched wild-type (WT) strain. We detected D in the polyA+ mRNA fraction from WT but not DUS KO (Fig 1A), confirming the hypothesis that DUS enzymes install D in mRNA. We therefore developed a method to map D at single nucleotide resolution by identifying chemical treatments that stall RT at D.

Methods
Synthetic RNAs for RNA degradation and RT stop testing
Synthetic 100% uridine or dihydrouridine containing RNA (5′-ggaacagaaacagagaaaggaacagagaaagacaU/DaaacagaaagagacaagaacagagacaagaacagU/DggcaggaacagagacaaacagagacaggaacaaU/DgacaggaacagaaagaaacagagacaagcacU/Dcgggcaccaaggacacgaaccggaacgcggaaccaaacgggcaacggaccggac-3′) was generated by run off transcription with T7 RNA polymerase and gel purified on an 8% urea-TBE polyacrylamide gel electrophoresis (PAGE) gel. To compare the harshness of the different D-modifying treatments, a synthetic RNA was incubated either under published D mapping conditions [6], under published D reduction conditions [21,22] or similar D reduction conditions with NaBH3CN substituted for NaBH4. To measure RT at reduced dihydrouridine, we reverse transcribed reduced U or D RNA with Superscript III RT, using manufacturer conditions. Samples were prepared and run on sequencing gels as in [37].

Citation: Draycott AS, Schaening-Burgos C, Rojas-Duran MF, Wilson L, Schärfen L, Neugebauer KM, et al. (2022) Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA. PLoS Biol 20(5): e3001622. https://doi.org/10.1371/journal.pbio.3001622

Academic Editor: Jeff Coller, Johns Hopkins University, UNITED STATES

Received: July 16, 2021; Accepted: April 7, 2022; Published: May 24, 2022

Copyright: © 2022 Draycott et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are found either in the paper and its Supporting Information files, or in GEO accession GSE175549. Yeast strains are available upon request.

Funding: Development of D-seq was supported by National Institute of Environmental Health Sciences (https://www.niehs.nih.gov/) grant 1R21ES031525 to WG, National Cancer Institute (https://www.cancer.gov/) grant 5R21CA246118 to WG, National Institute of General Medical Sciences (https://www.nigms.nih.gov/) grant 5R01GM112766 to KMN, William Raveis Charitable Fund Dale F. Frey Breakthrough Scientist Award DFS-34-19 from the Damon Runyon Foundation (https://www.damonrunyon.org/) to S.N. LS was supported by American Heart Association (https://www.heart.org/) grant 908949, AD was supported by National Cancer Institute (https://www.cancer.gov/) grant 1F31CA254339 and a Gruber Foundation Fellowship (https://gruber.yale.edu/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: D, dihydrouridine; DMS, dimethyl sulfate; D-seq, dihydrouridine sequencing; DUS, dihydrouridine synthase; KH, K homology domain; LC–MS, liquid chromatography–mass spectrometry; m1A, 1-methyladnosine; m2,2G, N2, N2-dimethylguanosine; m7G, 7-methylguanosine; miRNA, micro RNA; ncRNA, non-coding RNA; RRL, rabbit reticulocyte lysate; RRM, RNA recognition motif domain; RT, reverse transcriptase; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; UMI, unique molecular identifier; WT, wild-type

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001622#abstract0