Dataset Information

Chemoproteomic capture of RNA binding activity in living cells

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 >5,500 cysteine sites across ~3,000 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Dina Bai

LAB HEAD: Ku-Lung Hsu

PROVIDER: PXD044625 | Pride | 2023-10-10

REPOSITORIES: Pride

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			Action	DRS
	20191023_AH_PQE_Sol_InSitu_Pu1_DMSO_12_JWB1_20191031.raw	Raw
	20191023_AH_PQE_Sol_InSitu_Pu1_DMSO_12_JWB1_20191031.raw_20200116_Byonic_C.mgf	Mgf
	20191023_AH_PQE_Sol_InSitu_Pu1_DMSO_12_JWB1_20191031.raw_20200116_Byonic_C.mzid.gz	Mzid
	20191023_AH_PQE_Sol_InSitu_Pu1_Pu1_11_JWB1_20191031.raw	Raw
	20191023_AH_PQE_Sol_InSitu_Pu1_Pu1_11_JWB1_20191031.raw_20191104_Byonic_C.mgf	Mgf

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Chemoproteomic capture of RNA binding activity in living cells.

Heindel Andrew J AJ Brulet Jeffrey W JW Wang Xiantao X Founds Michael W MW Libby Adam H AH Bai Dina L DL Lemke Michael C MC Leace David M DM Harris Thurl E TE Hafner Markus M Hsu Ku-Lung KL

Nature communications 20231007 1

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 dire ...[more]

PMID: 37805600

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