Abstract
Mammalian cells express a wide range of transcripts, some protein-coding RNAs (mRNA) and many noncoding (nc) RNAs. Long (l)ncRNAscan modulates protein expression patterns by regulating gene transcription, pre-mRNA splicing, mRNA export, mRNA degradation, protein translation, and protein ubiquitination. Given the growing recognition that lncRNAs have a robust impact upon gene expression, there is rising interest in elucidating the levels and regulation of lncRNAs. A number of high-throughput methods have been developed recently to map the interaction of lncRNAs and RNA-binding proteins (RBPs). However, few of these approaches are suitable for mapping and quantifying RBP-lncRNA interactions. Here, we describe the recently developed method CLIP-qPCR (cross-linking and immunoprecipitation followed by reverse transcription and quantitative PCR) for mapping and quantifying RBP-lncRNA interactions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Berger SL (2002) Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12:142–148
Glisovic T, Bachorik JL, Yong J, Dreyfuss G (2008) RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett 582:1977–1986
Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102–114
Yoon JH, Abdelmohsen K, Gorospe M (2013) Posttranscriptional gene regulation by long noncoding RNA. J Mol Biol 425:3723–3730
Yoon JH, Abdelmohsen K, Gorospe M (2014) Functional interactions among microRNAs and long noncoding RNAs. Semin Cell Dev Biol 34:9–14
Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166
Riley KJ, Steitz JA (2013) The “Observer Effect” in genome-wide surveys of protein-RNA interactions. Mol Cell 49:601–604
Lin RJ (2008) RNA -protein interaction protocols. Methods Mol Biol 488:v–vii
Tenenbaum SA, Carson CC, Lager PJ, Keene JD (2000) Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A 97:14085–14090
Mukherjee N, Corcoran DL, Nusbaum JD, Reid DW, Georgiev S, Hafner M, Ascano M Jr, Tuschl T, Ohler U, Keene JD (2011) Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability. Mol Cell 43:327–339
Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302:1212–1215
Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB (2009) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469
Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479–486
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
König J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17:909–915
Kudla G, Granneman S, Hahn D, Beggs JD, Tollervey D (2011) Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast. Proc Natl Acad Sci U S A 108:10010–10015
Acknowledgments
JHY and MG were supported by the National Institute on Aging Intramural Research Program, National Institutes of Health and startup fund from Medical University of South Carolina.
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
Yoon, JH., Gorospe, M. (2016). Cross-Linking Immunoprecipitation and qPCR (CLIP-qPCR) Analysis to Map Interactions Between Long Noncoding RNAs and RNA-Binding Proteins. In: Feng, Y., Zhang, L. (eds) Long Non-Coding RNAs. Methods in Molecular Biology, vol 1402. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3378-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3378-5_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3376-1
Online ISBN: 978-1-4939-3378-5
eBook Packages: Springer Protocols