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

Nature Chemical Biology volume 17, pages 1178–1187 (2021)Cite this article

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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.

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Fig. 1: RNABPP enables the chemoproteomic analysis of RNA-modifying enzymes in living cells.
Fig. 2: Proteomic analysis of 5-FCyd-reactive proteins on mRNA using RNABPP.
Fig. 3: NSUN2 and TRMT2A are the major RNA m5C and m5U methyltransferases, respectively.
Fig. 4: DUS3L installs DHU on human RNA.
Fig. 5: 5-FUrd-iCLIP sequencing of DUS3L substrates.
Fig. 6: DUS3L regulates cell proliferation and protein translation efficiency.

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Data availability

The sequencing data reported in this paper have been deposited in the NCBI Gene Expression Omnibus (accession code GSE175825). The proteomics data reported in this paper are available via ProteomeXchange with identifier PXD022645. Source data are provided with this paper.

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Acknowledgements

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.

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Author notes

  1. These authors contributed equally: Wei Dai, Ang Li.

Authors and Affiliations

  1. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Wei Dai, Ang Li, Nathan J. Yu & Ralph E. Kleiner

  2. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Thao Nguyen & Martin Wühr

  3. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Thao Nguyen, Robert W. Leach & Martin Wühr

  4. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA

    Thao Nguyen

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Contributions

R.E.K. conceived the study, analyzed data, wrote the manuscript and performed experiments. W.D. performed RNABPP experiments, nucleoside MS, crosslinking studies and protein translation assays. A.L. performed iCLIP experiments and bioinformatic analysis. R.W.L. performed the bioinformatic analysis. N.J.Y. performed cell viability assays and crosslinking studies. T.N. performed MS proteomics and associated data analysis. M.W. supervised T.N.

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Correspondence to Ralph E. Kleiner.

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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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Supplementary information

Supplementary Information

Supplementary Tables 1–9 and Figs. 1–17.

Reporting Summary

Supplementary Data 1

LC–MS/MS proteomics data for Fig. 2a.

Supplementary Data 2

5-FUrd DUS3L iCLIP peak data.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

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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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