System-wide identification of RNA-binding proteins by interactome capture

Castello, A. , Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L. M., Krijgsveld, J. and Hentze, M. W. (2013) System-wide identification of RNA-binding proteins by interactome capture. Nature Protocols, 8(3), pp. 491-500. (doi: 10.1038/nprot.2013.020) (PMID:23411631)

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Abstract

Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Castello, Professor Alfredo
Authors: Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L. M., Krijgsveld, J., and Hentze, M. W.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Journal Name:Nature Protocols
Publisher:Nature Publishing Group
ISSN:1754-2189
ISSN (Online):1750-2799
Published Online:14 February 2013

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