Abstract
The importance of posttranscriptional regulation in cellular metabolism has recently gone beyond what was previously appreciated. The regulatory mechanisms are controlled by RNA-binding proteins (RBPs), which form complexes with RNA and regulate RNA processing, stability, and localization, among others. Consistently, mutations in RBPs result in defects in developmental processes, diseases, and cancer. Gaining deeper insights into the biology of RNA–RBP interactions will lead to a better understanding of regulatory processes and disease development. Several techniques have been developed to capture the properties of RNA–RBP interactions. Furthermore, the development of high-throughput sequencing has broadened the capability of these methods. Here, we summarize individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), a powerful technique that provides genome-wide information on RNA–RBP interactions at nucleotide resolution. In this chapter, we outline the iCLIP protocol and list possible controls that allow a targeted and cost-minimizing optimization of the protocol for an RBP-of-interest. Moreover, we provide notes on experimental design and a troubleshooting guideline for common problems that can occur during iCLIP library preparation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kaper JM (1969) Nucleic acid-protein interactions in turnip yellow mosaic virus. Science 166(3902):248–250
Gerstberger S, Hafner M, Tuschl T (2014) A census of human RNA-binding proteins. Nat Rev Genet 15:829–845
Greenberg JR (1979) Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res 6(2):715–732
Lunde BM, Moore C, Varani G (2007) RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8:479–490
Scherrer T, Mittal N, Janga SC et al (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 et al (2010) Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PLoS One 5:e12671
Castello A, Fischer B, Eichelbaum K et al (2012) Insights into RNA biology from an atlas of mammalian mRNA-Binding proteins. Cell 149:1393–1406
Kwon SC, Yi H, Eichelbaum K et al (2013) The RNA-binding protein repertoire of embryonic stem cells. Nat Struct Mol Biol 20:1122–1130
Baltz AG, Munschauer M, Schwanhäusser B et al (2012) The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46:674–690
Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136:777–793
Chelly J, Mandel JL (2001) Monogenic causes of X-linked mental retardation. Nat Rev Genet 2:669–680
Mili S, Steitz JA (2004) Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10:1692–1694
Ule J, Jensen K, Mele A et al (2005) CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37:376–386
Ule J, Jensen KB, Ruggiu M et al (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302:1212–1215
Hafner M, Landthaler M, Burger L et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141
Licatalosi DD, Mele A, Fak JJ et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469
Saulière J, Murigneux V, Wang Z et al (2012) CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex. Nat Struct Mol Biol 19:1124–1131
König J, Zarnack K, Rot G et al (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17:909–915
Sugimoto Y, König J, Hussain S et al (2012) Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions. Genome Biol 13:R67
Huppertz I, Attig J, D’Ambrogio A et al (2014) iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65:274–287
Jangi M, Boutz PL, Paul P et al (2014) Rbfox2 controls autoregulation in RNA-binding protein networks. Genes Dev 28:637–651
Zarnack K, König J, Tajnik M et al (2013) Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell 152:453–466
Hussain S, Sajini AA, Blanco S et al (2013) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4:255–261
Broughton JP, Pasquinelli AE (2013) Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP. Methods 63:119–125
Bosson AD, Zamudio JR, Sharp PA (2014) Endogenous miRNA and target concentrations determine susceptibility to potential ceRNA competition. Mol Cell 56:347–359
Acknowledgements
This protocol is based on previous versions that were derived with a lot of feedback and discussions in the Ule lab. We would also like to thank all iCLIP users for their feedback and comments that help to continuously improve the protocol. We acknowledge Dr. Kathi Zarnack and members of the König lab for discussions and help on the manuscript.
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
Sutandy, F.R., Hildebrandt, A., König, J. (2016). Profiling the Binding Sites of RNA-Binding Proteins with Nucleotide Resolution Using iCLIP. 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3067-8_11
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