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
Date Published2021 Nov
KeywordsCell Line, Humans, Oxidoreductases, RNA

<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>

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