Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation.

TitleActivity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation.
Publication TypeJournal Article
Year of Publication2021
AuthorsDai, W, Li, A, Yu, NJ, Nguyen, T, Leach, RW, Wühr, M, Kleiner, RE
JournalNat Chem Biol
Volume17
Issue11
Pagination1178-1187
Date Published2021 11
ISSN1552-4469
KeywordsCell Line, Humans, Oxidoreductases, RNA
Abstract

<p>Epitranscriptomic RNA modifications can regulate RNA activity; however, there remains a major gap in our understanding of the RNA chemistry present in biological systems. Here we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy that relies on metabolic RNA labeling, mRNA interactome capture and quantitative proteomics, to investigate RNA-modifying enzymes in human cells. RNABPP with 5-fluoropyrimidines allowed us to profile 5-methylcytidine (mC) and 5-methyluridine (mU) methyltransferases. Further, we uncover a new mechanism-based crosslink between 5-fluorouridine (5-FUrd)-modified RNA and the dihydrouridine synthase (DUS) homolog DUS3L. We investigate the mechanism of crosslinking and use quantitative nucleoside liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and 5-FUrd-based crosslinking and immunoprecipitation (CLIP) sequencing to map DUS3L-dependent dihydrouridine (DHU) modifications across the transcriptome. Finally, we show that DUS3L-knockout (KO) cells have compromised protein translation rates and impaired cellular proliferation. Taken together, our work provides a general approach for profiling RNA-modifying enzyme activity in living cells and reveals new pathways for epitranscriptomic RNA regulation.</p>

DOI10.1038/s41589-021-00874-8
Alternate JournalNat Chem Biol
PubMed ID34556860
PubMed Central IDPMC8551019
Grant ListR01 GM132189 / GM / NIGMS NIH HHS / United States
R35 GM128813 / GM / NIGMS NIH HHS / United States