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Post-Transcriptional Gene Regulation pp 153–173Cite as

PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1358))

Abstract

During post-transcriptional gene regulation (PTGR), RNA binding proteins (RBPs) interact with all classes of RNA to control RNA maturation, stability, transport, and translation. Here, we describe Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a transcriptome-scale method for identifying RBP binding sites on target RNAs with nucleotide-level resolution. This method is readily applicable to any protein directly contacting RNA, including RBPs that are predicted to bind in a sequence- or structure-dependent manner at discrete RNA recognition elements (RREs), and those that are thought to bind transiently, such as RNA polymerases or helicases.

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References

  1. Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136:731–745

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Moore MJ, Proudfoot NJ (2009) Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136:688–700

    Article  CAS  PubMed  Google Scholar 

  3. Martin KC, Ephrussi A (2009) mRNA localization: gene expression in the spatial dimension. Cell 136:719–730

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Cech TR, Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157:77–94

    Article  CAS  PubMed  Google Scholar 

  5. Lukong KE, Chang K-W, Khandjian EW, Richard S (2008) RNA-binding proteins in human genetic disease. Trends Genet 24:416–425

    Article  CAS  PubMed  Google Scholar 

  6. Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136:777–793

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Castello A, Fischer B, Hentze MW, Preiss T (2013) RNA-binding proteins in Mendelian disease. Trends Genet 29:318–327

    Article  CAS  PubMed  Google Scholar 

  8. Gerstberger S, Hafner M, Tuschl T (2014) A census of human RNA-binding proteins. Nat Rev Genet 15:829–845

    Article  CAS  PubMed  Google Scholar 

  9. König J, Zarnack K, Luscombe NM, Ule J (2011) Protein-RNA interactions: new genomic technologies and perspectives. Nat Publ Group 13:77–83

    Google Scholar 

  10. 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Gilbert C, Svejstrup JQ (2006) RNA immunoprecipitation for determining RNA-protein associations in vivo. Curr Protoc Mol Biol Chapter 27, Unit 27.4–27.4.11

    Google Scholar 

  12. Gerber AP, Luschnig S, Krasnow MA et al (2006) Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc Natl Acad Sci U S A 103:4487–4492

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. López de Silanes I, Zhan M, Lal A et al (2004) Identification of a target RNA motif for RNA-binding protein HuR. Proc Natl Acad Sci U S A 101:2987–2992

    Article  PubMed  Google Scholar 

  14. Maes OC, Chertkow HM, Wang E, Schipper HM (2009) MicroRNA: implications for Alzheimer disease and other human CNS disorders. Curr Genomics 10:154–168

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Hafner M, Renwick N, Farazi TA et al (2012) Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods 58:164–170

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Ascano M, Hafner M, Cekan P et al (2011) Identification of RNA-protein interaction networks using PAR-CLIP. WIREs Interdiscip Rev RNA 3:159–177

    Article  Google Scholar 

  18. Corcorabin DL, Georgiev S, Mukherjee N et al (2011) PARalyzer: definition of RNAding sites from PAR-CLIP short-read sequence data. Genome Biol 12:R79

    Article  Google Scholar 

  19. Chen B, Yun J, Kim MS et al (2014) PIPE-CLIP: a comprehensive online tool for CLIP-seq data analysis. Genome Biol 15:1–10

    Article  Google Scholar 

  20. Sievers C, Schlumpf T, Sawarkar R, Comoglio F, Paro R (2012) Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data. Nucleic Acids Res 40(2):160

    Article  Google Scholar 

  21. Anders G, Mackowiak SD, Jens M et al (2012) doRiNA: a database of RNA interactions in post-transcriptional regulation. Nucleic Acids Res 40:D180–D186

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Khorshid M, Rodak C, Zavolan M (2011) CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins. Nucleic Acids Res 39:D245–D252

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Yang JH, Li JH, Shao P et al (2011) starBase: a database for exploring microRNA-mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data. Nucleic Acids Res 39:D202–D209

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Chou CH, Lin FM, Chou MT et al (2013) A computational approach for identifying microRNA-target interactions using high-throughput CLIP and PAR-CLIP sequencing. BMC Genomics 14(Suppl 1):S2

    PubMed Central  PubMed  Google Scholar 

  25. Uren PJ, Bahrami-Samani E, Burns SC et al (2012) Site identification in high-throughput RNA-protein interaction data. Bioinformatics 28:3013–3020

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Wang T, Xie Y, Xiao G (2014) dCLIP: a computational approach for comparative CLIP-seq analyses. Genome Biol 15:R11

    Article  PubMed Central  PubMed  Google Scholar 

  27. Bailey TL (2002) Discovering novel sequence motifs with MEME. Curr Protoc Bioinformatics Chapter 2, Unit 2.4–2.4.35

    Google Scholar 

  28. Liu XS, Brutlag DL, Liu JS (2002) An algorithm for finding protein–DNA binding sites with applications to chromatin-immunoprecipitation microarray experiments. Nat Biotechnol 20:835–839

    Article  CAS  PubMed  Google Scholar 

  29. Siddharthan R, Siggia ED, van Nimwegen E (2005) PhyloGibbs: a Gibbs sampling motif finder that incorporates phylogeny. PLoS Comput Biol 1:e67

    Article  PubMed Central  PubMed  Google Scholar 

  30. Georgiev S, Boyle AP, Jayasurya K et al (2010) Evidence-ranked motif identification. Genome Biol 11:R19

    Article  PubMed Central  PubMed  Google Scholar 

  31. Ng P, Keich U (2008) GIMSAN: a Gibbs motif finder with significance analysis. Bioinformatics 24:2256–2257

    Article  CAS  PubMed  Google Scholar 

  32. Brewis IA, Brennan P (2010) Proteomics technologies for the global identification and quantification of proteins. Adv Protein Chem Struct Biol 80:1–44

    Article  CAS  PubMed  Google Scholar 

  33. Guruharsha KG, Rual JF, Zhai B et al (2011) A protein complex network of Drosophila melanogaster. Cell 147:690–703

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Kucerova L, Poturnajova M, Tyciakova S, Matuskova M (2012) Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase. Stem Cell Res 8:247–258

    Article  CAS  PubMed  Google Scholar 

  35. Jungkamp AC, Stoeckius M, Mecenas D et al (2011) In vivo and transcriptome-wide identification of RNA binding protein target sites. Mol Cell 44:828–840

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS / NIH, 50 South Drive, 20892, Bethesda, MD, USA

    Charles Danan, Sudhir Manickavel & Markus Hafner

Authors
  1. Charles Danan
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  2. Sudhir Manickavel
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  3. Markus Hafner
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Corresponding author

Correspondence to Markus Hafner .

Editor information

Editors and Affiliations

  1. Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento, Mattarello, Trento, Italy

    Erik Dassi

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Danan, C., Manickavel, S., Hafner, M. (2016). PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites. 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_10

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  • DOI: https://doi.org/10.1007/978-1-4939-3067-8_10

  • 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

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