Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation. Author Wei Dai, Ang Li, Nathan Yu, Thao Nguyen, Robert Leach, Martin Wühr, Ralph Kleiner Publication Year 2021 Type Journal Article Abstract 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. Keywords Humans, Cell Line, RNA, Oxidoreductases Journal Nat Chem Biol Volume 17 Issue 11 Pages 1178-1187 Date Published 2021 Nov ISSN Number 1552-4469 DOI 10.1038/s41589-021-00874-8 Alternate Journal Nat Chem Biol PMCID PMC8551019 PMID 34556860 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML