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 (m5C) and 5-methyluridine (m5U) 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.
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We thank C. DeCoste and K. Rittenbach at the Princeton University Flow Cytometry Resource Facility for assistance with flow cytometry analysis. We thank L. Ryazanova (supported by the Princeton Catalysis Initiative and the Lewis–Sigler Collaboration Fund) for technical assistance. R.E.K. acknowledges support from a National Science Foundation CAREER award (MCB-1942565), the National Institute of Health (R01GM132189), the Sidney Kimmel Foundation and the Alfred P. Sloan Foundation. This work was supported by NIH grant R35 GM128813 (to M.W.). T.N. was supported by the American Heart Association. W.D. was generously supported by the Edward C. Taylor 3rd Year Graduate Fellowship in Chemistry. A.L. was supported by the Princeton Catalysis Initiative. All authors thank Princeton University for financial support.
The authors declare no competing financial interests.
Peer review information Nature Chemical Biology thanks Jing Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Dai, W., Li, A., Yu, N.J. et al. Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation. Nat Chem Biol 17, 1178–1187 (2021). https://doi.org/10.1038/s41589-021-00874-8
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