Cross-Linking Immunoprecipitation and qPCR (CLIP-qPCR) Analysis to Map Interactions Between Long Noncoding RNAs and RNA-Binding Proteins

  • Je-Hyun YoonEmail author
  • Myriam Gorospe
Part of the Methods in Molecular Biology book series (MIMB, volume 1402)


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.

Key words

CLIP lncRNA RBP Ribonucleoprotein complexes qPCR 



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.


  1. 1.
    Berger SL (2002) Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12:142–148CrossRefPubMedGoogle Scholar
  2. 2.
    Glisovic T, Bachorik JL, Yong J, Dreyfuss G (2008) RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett 582:1977–1986CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102–114CrossRefPubMedGoogle Scholar
  4. 4.
    Yoon JH, Abdelmohsen K, Gorospe M (2013) Posttranscriptional gene regulation by long noncoding RNA. J Mol Biol 425:3723–3730CrossRefPubMedGoogle Scholar
  5. 5.
    Yoon JH, Abdelmohsen K, Gorospe M (2014) Functional interactions among microRNAs and long noncoding RNAs. Semin Cell Dev Biol 34:9–14CrossRefPubMedGoogle Scholar
  6. 6.
    Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166CrossRefPubMedGoogle Scholar
  7. 7.
    Riley KJ, Steitz JA (2013) The “Observer Effect” in genome-wide surveys of protein-RNA interactions. Mol Cell 49:601–604CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lin RJ (2008) RNA -protein interaction protocols. Methods Mol Biol 488:v–viiPubMedGoogle Scholar
  9. 9.
    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 97:14085–14090CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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 43:327–339CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB (2003) CLIP identifies Nova-regulated RNA networks in the brain. Science 302:1212–1215CrossRefPubMedGoogle Scholar
  12. 12.
    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 456:464–469CrossRefGoogle Scholar
  13. 13.
    Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479–486PubMedPubMedCentralGoogle Scholar
  14. 14.
    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 141:129–141CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    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 17:909–915CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    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 108:10010–10015CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyMedical University of South CarolinaCharlestonUSA
  2. 2.Laboratory of Genetics and GenomicsNational Institute on Aging-Intramural Research Program, National Institutes of HealthBaltimoreUSA

Personalised recommendations