Epitranscriptomic RNA modifications can regulate RNA activity, however there remains a major gap in our understanding of the scope of RNA chemistry present in biological systems, the enzymes responsible for regulating modification levels, and the ultimate function of these modifications in biological processes. To address this gap, we have developed RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy relying upon metabolic RNA labeling with modified nucleoside probes, RNA-protein enrichment, and quantitative proteomics. We apply RNABPP with fluoropyrimidine nucleosides in human cells in order to investigate RNA pyrimidine-modifying enzymes acting upon mRNA. In addition to profiling 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferase activity on mRNA, we identify a novel mechanism-based RNA-protein crosslink between 5-fluorouridine-modified RNA and the uncharacterized human dihydrouridine synthase DUS3L. Further, we study the cellular substrates of DUS3L using quantitative nucleoside LC-MS/MS and catalytic cross-linking and immunoprecipitation-based sequencing. Taken together, our work provides a general approach for profiling RNA modifying enzyme activity in vivo, and reveals the existence of new pathways for the epitranscriptomic regulation of RNA behavior in human cells.
Support or Funding Information
NIH R01 GM132189
NSF CAREER Award MCB-1942565
lt;pgt;NIH R01 GM132189lt;/pgt;lt;pgt;NSF CAREER Awardamp;nbsp;MCB-1942565lt;/pgt;
