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.
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Acknowledgements
We thank M. Landthaler (Max Delbrück Center for Molecular Medicine) for generously sharing his expertise on PAR-CL. We are grateful to R. Pepperkok (EMBL) for plasmids and M. Gromeier (Duke University Medical Center) for the HeLa Flip-in TRex cell line. We thank M. Muckenthaler (University of Heidelberg) for Huh-7 cells. We acknowledge A. Perez and the EMBL Flow Cytometry Core Facility for FACS experiments, and EMBL Gene and Proteomics Core Facilities for support throughout this work. We also thank Chromotek for expert technical advice and support. A.C. is the beneficiary of a Marie Curie postdoctoral fellowship (FP7). M.W.H. acknowledges support by the European Research Council (ERC) Advanced Grant ERC-2011-ADG_20110310 and the Virtual Liver Network of the German Ministry for Science and Education.
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A.C., J.K., L.M.S. and M.W.H. contributed to the conception and design of the project. A.C., R.H., C.S., B.F. and K.E. carried out the experimental work. A.C. and M.W.H. wrote the manuscript.
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Castello, A., Horos, R., Strein, C. et al. System-wide identification of RNA-binding proteins by interactome capture. Nat Protoc 8, 491–500 (2013). https://doi.org/10.1038/nprot.2013.020
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DOI: https://doi.org/10.1038/nprot.2013.020
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