Abstract
RNA associates with RNA-binding proteins (RBPs) from synthesis to decay, forming dynamic ribonucleoproteins (RNPs). In spite of the preeminent role of RBPs regulating RNA fate, the scope of cellular RBPs has remained largely unknown. We have recently developed a novel and comprehensive method to identify the repertoire of active RBPs of cultured cells, called RNA interactome capture. Using in vivo UV cross-linking on cultured cells, proteins are covalently bound to RNA if the contact between the two is direct (“zero distance”). Protein-RNA complexes are purified by poly(A) tail-dependent oligo(dT) capture and analyzed by quantitative mass spectrometry. Because UV irradiation is applied to living cells and purification is performed using highly stringent washes, RNA interactome capture identifies physiologic and direct protein-RNA interactions. Applied to HeLa cells, this protocol revealed the near-complete repertoire of RBPs, including hundreds of novel RNA binders. Apart from its RBP discovery capacity, quantitative and comparative RNA interactome capture can also be used to study the responses of the RBP repertoire to different physiological cues and processes, including metabolic stress, differentiation, development, or the response to drugs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Scherrer T, Mittal N, Janga SC, Gerber AP (2010) A screen for RNA-binding proteins in yeast indicates dual functions for many enzymes. PLoS One 5, e15499
Tsvetanova NG, Klass DM, Salzman J, Brown PO (2010) Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PLoS One 5:e12671, 12610.11371/journal.pone.0012671
Anantharaman V, Koonin EV, Aravind L (2002) Comparative genomics and evolution of proteins involved in RNA metabolism. Nucleic Acids Res 30:1427–1464
Castello A, Fischer B, Eichelbaum K, Horos R, Beckmann BM, Strein C, Davey NE, Humphreys DT, Preiss T, Steinmetz LM, Krijgsveld J, Hentze MW (2012) Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149:1393–1406
Baltz AG, Munschauer M, Schwanhausser B, Vasile A, Murakawa Y, Schueler M, Youngs N, Penfold-Brown D, Drew K, Milek M, Wyler E, Bonneau R, Selbach M, Dieterich C, Landthaler M (2012) The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46:674–690
Kwon SC, Yi H, Eichelbaum K, Fohr S, Fischer B, You KT, Castello A, Krijgsveld J, Hentze MW, Kim VN (2013) The RNA-binding protein repertoire of embryonic stem cells. Nat Struct Mol Biol 20:1122–1130
Mitchell SF, Jain S, She M, Parker R (2013) Global analysis of yeast mRNPs. Nat Struct Mol Biol 20:127–133
Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141
Pashev IG, Dimitrov SI, Angelov D (1991) Crosslinking proteins to nucleic acids by ultraviolet laser irradiation. Trends Biochem Sci 16:323–326
Suchanek M, Radzikowska A, Thiele C (2005) Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat Methods 2:261–267
Castello A, Horos R, Strein C, Fischer B, Eichelbaum K, Steinmetz LM, Krijgsveld J, Hentze MW (2013) System-wide identification of RNA-binding proteins by interactome capture. Nat Protoc 8:491–500
Strein C, Alleaume AM, Rothbauer U, Hentze MW, Castello A (2014) A versatile assay for RNA-binding proteins in living cells. RNA 20:721–731
Acknowledgements
A.C. acknowledges support by the MRC Career Development Award Ref: MR/L019434/1. M.W.H. acknowledges support by the ERC Advanced Grant ERC-2011-ADG_20110310 and by a grant from the Virtual Liver Network of the German Ministry for Science and Education. A.C. is funded by the MRC Career Development Award number MR/L019434/1
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Castello, A. et al. (2016). Comprehensive Identification of RNA-Binding Proteins by RNA Interactome Capture. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 1358. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3067-8_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3067-8_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3066-1
Online ISBN: 978-1-4939-3067-8
eBook Packages: Springer Protocols