Streamlined Purification of RNA-Protein Complexes Using UV Cross-Linking and RNA Antisense Purification

RNA-protein interactions are important in development and disease, but identification of novel RNA-protein interactions remains challenging. Here, we describe an updated capture method to identify direct and specific RNA-protein interactions. First, RNA and protein are covalently cross-linked in liv...

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Bibliographic Details
Published in:	Methods in molecular biology (Clifton, N.J.) Vol. 2666; p. 213
Main Authors:	Trang, Nhu, Su, Tong, Hall, Simone, Boutros, Nada, Kong, Bobby, Huang, Calvin, McHugh, Colleen A
Format:	Journal Article
Language:	English
Published:	United States 2023
Subjects:	DNA - metabolism Nucleic Acid Hybridization - methods Proteins - genetics RNA - genetics Ultraviolet Rays UV cross-linking Nucleic acid biology RNA–protein interactions Endogenous complexes Mass spectrometry Macromolecular complex purification
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Summary:	RNA-protein interactions are important in development and disease, but identification of novel RNA-protein interactions remains challenging. Here, we describe an updated capture method to identify direct and specific RNA-protein interactions. First, RNA and protein are covalently cross-linked in living cells by treatment with UV light at 254 nanometers wavelength. The antisense purification approach is dependent upon nucleic acid hybridization between biotinylated DNA probes and a target RNA. Target protein:RNA:DNA complexes are enriched by capture on streptavidin magnetic beads and purified through several denaturing washes that remove nonspecific protein and nucleic acid interactors. Mass spectrometry is used to identify proteins that are specifically enriched in the target RNA capture. This method has been applied to discover the protein interactions of noncoding RNAs but can be used to capture any RNA where the target sequence is known.
ISSN:	1940-6029
DOI:	10.1007/978-1-0716-3191-1_16