Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation

  • Nat Chem Biol. 2021 Nov;17(11):1178-1187. doi: 10.1038/s41589-021-00874-8.
Wei Dai  #  1 ,  Ang Li  #  1 ,  Nathan J Yu  1 ,  Thao Nguyen  2  3  4 ,  Robert W Leach  3 ,  Martin Wühr  2  3 ,  Ralph E Kleiner  5
Affiliations
  • 1. Department of Chemistry, Princeton University, Princeton, NJ, USA.
  • 2. Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
  • 3. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA.
  • 4. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA.
  • 5. Department of Chemistry, Princeton University, Princeton, NJ, USA. [email protected].
  • # Contributed equally.
PMID: 34556860 DOI: 10.1038/s41589-021-00874-8
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 (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.