Chemoproteomic capture of RNA binding activity in living cells

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 and Ku-Lung Hsu ()
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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
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

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-41844-z

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