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

Wei Dai, Ang Li, Nathan J. Yu, Thao Nguyen, Robert W. Leach, Martin Wühr, Ralph E. Kleiner

Research output: Contribution to journalArticlepeer-review

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. [Figure not available: see fulltext.].

Original languageEnglish (US)
JournalNature Chemical Biology
DOIs
StateAccepted/In press - 2021

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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