Title | Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation. |
Publication Type | Journal Article |
Year of Publication | 2021 |
Authors | Dai, W, Li, A, Yu, NJ, Nguyen, T, Leach, RW, Wühr, M, Kleiner, RE |
Journal | Nat Chem Biol |
Volume | 17 |
Issue | 11 |
Pagination | 1178-1187 |
Date Published | 2021 Nov |
ISSN | 1552-4469 |
Keywords | Cell 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> |
DOI | 10.1038/s41589-021-00874-8 |
Alternate Journal | Nat Chem Biol |
PubMed ID | 34556860 |
PubMed Central ID | PMC8551019 |
Grant List | R01 GM132189 / GM / NIGMS NIH HHS / United States R35 GM128813 / GM / NIGMS NIH HHS / United States |