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nRIP-seq: A Technique to Identify RNA Targets of an RNA Binding Protein on a Genome-Wide Scale

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Regulatory Non-Coding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1206))

Abstract

Native RNA immunoprecipitation (nRIP) coupled with high-throughput sequencing (nRIP-seq) is a powerful technique that allows transcriptome-wide identification of the entire subset of coding and noncoding RNAs associated with a particular protein. Since this technology is carried out in a native condition without cross-linking, nRIP-seq detects RNAs that bind a protein directly or indirectly through a larger RNA–protein complex. Here, we use the interaction between RNA and chromatin modifiers, Polycomb proteins, as an example to describe this method. Using nRIP-seq, we provide a snapshot of Ezh2, a Polycomb component, and RNA interaction in mouse embryonic stem cells.

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References

  1. Claverie JM (2005) Fewer genes, more noncoding RNA. Science 309:1529–1530

    Article  PubMed  CAS  Google Scholar 

  2. Carninci P et al (2005) The transcriptional landscape of the mammalian genome. Science 2309:1559–1563

    Google Scholar 

  3. Czech B, Hannon GJ (2011) Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 12:19–31

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43:904–914

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Guttman M et al (2011) lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477:295–300

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Lanz RB et al (1999) A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97:17–27

    Article  PubMed  CAS  Google Scholar 

  7. Willingham AT et al (2005) A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science 309: 1570–1573

    Article  PubMed  CAS  Google Scholar 

  8. Rinn JL et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1323

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Zhao J et al (2008) Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322:750–756

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Pandey RR et al (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 32:232–246

    Article  PubMed  CAS  Google Scholar 

  11. Nagano T et al (2008) The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322: 1717–1720

    Article  PubMed  CAS  Google Scholar 

  12. Bertani S et al (2011) The noncoding RNA Mistral activates Hoxa6 and Hoxa7 expression and stem cell differentiation by recruiting MLL1 to chromatin. Mol Cell 43:1040–1046

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Chen LL, Carmichael GG (2009) Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA. Mol Cell 35:467–478

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. Clemson CM et al (2009) An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell 33:717–726

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Zhao J et al (2010) Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell 40:939–953

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  16. Keene JD, Komisarow JM, Friedersdorf MB (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc 1:302–307

    Article  PubMed  CAS  Google Scholar 

  17. Cloonan N et al (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5:613–619

    Article  PubMed  CAS  Google Scholar 

  18. Matz M et al (1999) Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res 27: 1558–1560

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Pinto FL, Lindblad P (2010) A guide for in-house design of template-switch-based 5′ rapid amplification of cDNA ends systems. Anal Biochem 397:227–232

    Article  PubMed  Google Scholar 

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Correspondence to Jing Crystal Zhao .

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Zhao, J.C. (2015). nRIP-seq: A Technique to Identify RNA Targets of an RNA Binding Protein on a Genome-Wide Scale. In: Carmichael, G. (eds) Regulatory Non-Coding RNAs. Methods in Molecular Biology, vol 1206. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1369-5_9

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  • DOI: https://doi.org/10.1007/978-1-4939-1369-5_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1368-8

  • Online ISBN: 978-1-4939-1369-5

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