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Chemoproteomic capture of RNA binding activity in living cells

Author

Listed:
  • Andrew J. Heindel

    (University of Virginia School of Medicine)

  • Jeffrey W. Brulet

    (University of Virginia)

  • Xiantao Wang

    (RNA Molecular Biology Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Disease)

  • Michael W. Founds

    (University of Virginia)

  • Adam H. Libby

    (University of Virginia
    University of Virginia Cancer Center, University of Virginia)

  • Dina L. Bai

    (University of Virginia)

  • Michael C. Lemke

    (University of Virginia School of Medicine)

  • David M. Leace

    (University of Virginia School of Medicine)

  • Thurl E. Harris

    (University of Virginia School of Medicine)

  • Markus Hafner

    (RNA Molecular Biology Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Disease)

  • Ku-Lung Hsu

    (University of Virginia School of Medicine
    University of Virginia
    University of Virginia Cancer Center, University of Virginia
    University of Virginia)

Abstract

Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.

Suggested Citation

  • Andrew J. Heindel & Jeffrey W. Brulet & Xiantao Wang & Michael W. Founds & Adam H. Libby & Dina L. Bai & Michael C. Lemke & David M. Leace & Thurl E. Harris & Markus Hafner & Ku-Lung Hsu, 2023. "Chemoproteomic capture of RNA binding activity in living cells," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-41844-z
    DOI: 10.1038/s41467-023-41844-z
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    References listed on IDEAS

    as
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