Skip to main content
SpringerLink
Log in

RNA immunoprecipitation technique for Drosophila melanogaster S2 cells

  • Molecular Cell Biology
  • Published:
Molecular Biology Aims and scope Submit manuscript

Abstract

RNA-binding proteins play an important role in RNA metabolism, especially in mRNA biogenesis and subsequent expression patterns regulation. RNA immunoprecipitation (RIP) is a powerful tool for detecting protein–RNA associations. In this paper, we briefly cover the history of this method for analyzing RNA–protein interactions and reviewing a number of modifications of the RIP technique. We also present an adjusted RIP protocol that was modified for Drosophila S2 cell culture. The use of this protocol allows one to perform the efficient precipitation of RNA–protein complexes and harvest RNA in amounts that are sufficient for its downstream analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

RIP:

RNA immunoprecipitation

RNP:

ribonucleoprotein complex

RBP:

RNA-binding protein

CLIP:

the method of successive RNA–protein crosslinking and immunoprecipitation

HITS-CLIP:

high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation

References

  1. Clancy C., Brown W. 2008. Translation: DNA to mRNA to protein. Nat. Educat. 1, 101.

    Google Scholar 

  2. Moore M.J. 2005. From birth to death: The complex lives of eukaryotic mRNAs. Science. 309, 1514–1518.

    Article  CAS  PubMed  Google Scholar 

  3. Keene J.D. 2001. Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome. Proc. Natl. Acad. Sci. U. S. A. 98, 7018–7024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tian B., Bevilacqua P.C., Diegelman-Parente A., Mathews M.B. 2004. The double-stranded-RNAbinding motif: Interference and much more. Nat. Rev. Mol. Cell Biol. 5, 1013–1023.

    Article  CAS  PubMed  Google Scholar 

  5. Niranjanakumari S., Lasda E., Brazas R., Garcia-Blanco M.A. 2002. Reversible cross-linking combined with immunoprecipitation to study RNA–protein interactions in vivo. Methods. 26, 182–190.

    Article  CAS  PubMed  Google Scholar 

  6. Baroni T.E., Chittur S.V., George A.D., Tenenbaum S.A. 2008. Advances in RIP-chip analysis: RNA-binding protein immunoprecipitation-microarray profiling. Methods Mol. Biol. 419, 93–108.

    Article  CAS  PubMed  Google Scholar 

  7. Keene J.D., Tenenbaum S. 2002. Eukaryotic mRNPs may represent posttranscriptional operons. Mol. Cell. 9, 1161–1167.

    Article  CAS  PubMed  Google Scholar 

  8. Hieronymus H., Silver P.A. 2003. Genome-wide analysis of RNA–protein interactions illustrates specificity of the mRNA export machinery. Nat. Genet. 33, 155–161.

    Article  CAS  PubMed  Google Scholar 

  9. Popova V.V., Kurshakova M.M., Kopytova D.V. 2015. Methods to study the RNA–protein interactions. Mol. Biol. (Moscow). 49 (3), 418–426.

    Article  CAS  Google Scholar 

  10. Cilley C.D., Williamson J.R. 1999. PACE analysis of RNA–peptide interactions. Methods Mol. Biol. 118, 129–141.

    CAS  PubMed  Google Scholar 

  11. Amstutz P., Forrer P., Zahnd C., Pluckthun A. 2001. In vitro display technologies: Novel developments and applications. Curr. Opin. Biotechnol. 12, 400–405.

    Article  CAS  PubMed  Google Scholar 

  12. Bernstein D.S., Buter N., Stumpf C., Wickens M. 2002. Analyzing mRNA–protein complexes using a yeast three-hybrid system. Methods. 26, 123–141.

    Article  CAS  PubMed  Google Scholar 

  13. Sen Gupta D.J., Zhang B., Kraemer B., Pochart P., Fields S., Wickens M. 1996. A three-hybrid system to detect RNA–protein interactions in vivo. Proc. Natl. Acad. Sci. U. S. A. 93, 8496–8501.

    Article  CAS  Google Scholar 

  14. Wang Z.F., Whitfield M.L., Ingledue T.C., Dominski Z., Marzluff W.F. 1996. The protein that binds the 3' end of histone mRNA: A novel RNA-binding protein required for histone pre-mRNA processing. Genes Dev. 10, 3028–3040.

    Article  CAS  PubMed  Google Scholar 

  15. Chang J.S., Tan L., Schedl P. 1999. The Drosophila CPEB homolog, orb, is required for oskar protein expression in oocytes. Dev. Biol. 215, 91–106.

    Article  CAS  PubMed  Google Scholar 

  16. Deshpande G., Samuels M.E., Schedl P.D. 1996. Sexlethal interacts with splicing factors in vitro and in vivo. Mol. Cell. Biol. 16, 5036–5047.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tenenbaum S.A., Carson C.C., Lager PJ., Keene J.D. 2000. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc. Natl. Acad. Sci. U. S. A. 97, 14085–14090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Inada M., Guthrie C. 2004. Identification of Lhp1passociated RNAs by microarray analysis in Saccharomyces cerevisiae reveals association with coding and noncoding RNAs. Proc. Natl. Acad. Sci. U. S. A. 101, 434–439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gerber A.P., Luschnig S., Krasnow M.A., Brown P.O., Herschlag D. 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  CAS  PubMed  PubMed Central  Google Scholar 

  20. Roy P.J., Stuart J.M., Lund J., Kim S.K. 2002. Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature. 418, 975–979.

    CAS  PubMed  Google Scholar 

  21. Schmitz-Linneweber C., Williams-Carrier R., Barkan A. 2005. RNA immunoprecipitation and microarray analysis show a chloroplast Pentatricopeptide repeat protein to be associated with the 5' region of mRNAs whose translation it activates. Plant Cell. 17, 2791–2804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Galgano A., Forrer M., Jaskiewicz L., Kanitz A., Zavolan M., Gerber A.P. 2008. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system. PLoS ONE. 3, e3164.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Penalva L.O., Tenenbaum S.A., Keene J.D. 2004. Gene expression analysis of messenger RNP complexes. Methods Mol. Biol. 257, 125–134.

    CAS  PubMed  Google Scholar 

  24. Mili S., Steitz J.A. 2004. Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses. RNA. 10, 1692–1694.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cho Y.S., Iguchi N., Yang J., Handel M.A., Hecht N.B. 2005. Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis. Biol. Reprod. 73, 840–847.

    Article  CAS  PubMed  Google Scholar 

  26. Ule J., Jensen K.B., Ruggiu M., Mele A., Ule A., Darnell R.B. 2003. CLIP identifies Nova-regulated RNA networks in the brain. Science. 302, 1212–1215.

    Article  CAS  PubMed  Google Scholar 

  27. Brimacombe R., Stiege W., Kyriatsoulis A., Maly P. 1988. Intra-RNA and RNA–protein cross-linking techniques in Escherichia coli ribosomes. Methods Enzymol. 164, 287–309.

    Article  CAS  PubMed  Google Scholar 

  28. Darnell R.B. 2010. HITS-CLIP: Panoramic views of protein-RNA regulation in living cells. Wiley Interdiscip. Rev. RNA. 1, 266–286.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ascano M., Hafner M., Cekan P., Gerstberger S., Tuschl T. 2012. Identification of RNA–protein interaction networks using PAR-CLIP. Wiley Interdiscip. Rev. RNA. 3, 159–177.

    Article  CAS  PubMed  Google Scholar 

  30. Hafner M., Landthaler M., Burger L., Khorshid M., Hausser J., Berninger P., Rothballer A., Ascano M. Jr., Jungkamp A.C., Munschauer M., Ulrich A., Wardle G.S., Dewell S., Zavolan M., Tuschl T. 2010. Transcriptomewide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell. 141, 129–141.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Konig J., Zarnack K., Rot G., Curk T., Kayikci M., Zupan B., Turner D.J., Luscombe N.M., Ule J. 2011. iCLIP: Transcriptome-wide mapping of protein–RNA interactions with individual nucleotide resolution. J. Vis. Exp. 50, 1–7.

    Google Scholar 

  32. Hussain S., Sajini A.A., Blanco S., Dietmann S., Lombard P., Sugimoto Y., Paramor M., Gleeson J.G., Odom D.T., Ule J., Frye M. 2013. NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep. 4, 255–261.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Selth L.A., Gilbert C., Svejstrup J.Q. 2009. RNA immunoprecipitation to determine RNA–protein associations in vivo. Cold Spring Harb. Protoc. 4, 1–7.

    Google Scholar 

  34. Penalva L.O., Burdick M.D., Lin S.M., Sutterluety H., Keene J.D. 2004. RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells. Mol. Cancer. 3, 24.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Zang Z., Edenberg H.J., Davis R.L. 2005. Isolation of mRNA from specific tissues of Drosophilia by mRNA tagging. Nucl. Acids Res. 33, e148.

    Article  Google Scholar 

  36. Keene J.D., Komisarow J.M., Friedersdorf M.B. 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  CAS  PubMed  Google Scholar 

  37. Halbeisen R.E., Gerber A.P. 2009. Stress-dependent coordination of transcriptome and translatome in yeast. PLoS Biol. 7, e1000105.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Heiman M., Schaefer A., Gong S., Peterson J.D., Day M., Ramsey K.E., Suarez-Farinas M., Schwarz C., Stephan D.A., Surmeier D.J., Greengard P., Heintz N. 2008. A translational profiling approach for the molecular characterization of CNS cell types. Cell. 135, 738–748.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kunitomo H., Uesugi H., Kohara Y., Iino Y. 2005. Identification of ciliated sensory neuron-expressed genes in Caenorhabditis elegans using targeted pulldown of poly(A) tails. Genome Biol. 6, R17.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Azuma-Mukai A., Oguri H., Mituyama T., Qian Z.R., Asai K., Siomi H., Siomi M.C. 2008. Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing. Proc. Natl. Acad. Sci. U. S. A. 105, 7964–7969.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Karginov F.V., Conaco C., Xuan Z., Schmidt B.H., Parker J.S., Mandel G., Hannon G.J. 2007. A biochemical approach to identifying microRNA targets. Proc. Natl. Acad. Sci. U. S. A. 104, 19291–19296.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Landthaler M., Gaidatzis D., Rothballer A., Chen P.Y., Soll S.J., Dinic L., Ojo T., Hafner M., Zavolan M., Tuschl T. 2008. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA. 14, 2580–2596.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Jain R., Devine T., George A.D., Chittur S.V., Baroni T.E., Penalva L.O., Tenenbaum S.A. 2011. RIP-Chip analysis: RNA-binding protein immunoprecipitationmicroarray (Chip) profiling. Methods Mol. Biol. 703, 247–263.

    Article  CAS  PubMed  Google Scholar 

  44. Churchman L.S., Weissman J.S. 2011. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature. 469, 368–373.

    Article  CAS  PubMed  Google Scholar 

  45. Nojima T., Gomes T., Grosso A.R., Kimura H., Dye M.J., Dhir S., Carmo-Fonseca M., Proudfoot N.J. 2015. Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell. 161, 526–540.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Core L.J., Waterfall J.J., Lis J.T. 2008. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. 322, 1845–1848.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kwak H., Fuda N.J., Core L.J., Lis J.T. 2013. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science. 339, 950–953.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Cho J., Chang H., Kwon S.C., Kim B., Kim Y., Choe J., Ha M., Kim Y.K., Kim V.N. 2012. LIN28A is a suppressor of ER-associated translation in embryonic stem cells. Cell. 151, 765–777.

    Article  CAS  PubMed  Google Scholar 

  49. Hammell M., Long D., Zhang L., Lee A., Carmack C.S., Han M., Ding Y., Ambros V. 2008. mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts. Nat. Methods. 5, 813–819.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Fujiwara H., Ishikawa H. 1986. Molecular mechanism of introduction of the hidden break into the 28S rRNA of insects: Implication based on structural studies. Nucleic Acids Res. 14, 6393–6401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Winnebeck E.C., Millar C.D., Warman G.R. 2010. Why does insect RNA look degraded? J. Insect. Sci. 10, 159.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Moretti F., Kaiser C., Zdanowicz-Specht A., Hentze M.W. 2012. PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding. Nat. Struct. Mol. Biol. 19, 603–608.

    Article  CAS  PubMed  Google Scholar 

  53. Sachs A. 2000. Physical and functional interactions between the mRNA cap structure and the poly(A) tail. In: Translational Control of Gene Expression. Eds. Sonenberg N., Hershey J.W.B., Matthews M.B. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press, pp. 447–466.

    Google Scholar 

  54. Hosoda N.L.F., Maquat L.E. 2006. Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails. Mol. Cell. Biol. 26, 3085–3097.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia

    Z. M. Kachaev, R. A. Gilmutdinov, D. V. Kopytova, A. A. Zheludkevich, Y. V. Shidlovskii & A. S. Kurbidaeva

Authors
  1. Z. M. Kachaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. A. Gilmutdinov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. V. Kopytova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. A. Zheludkevich
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Y. V. Shidlovskii
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. S. Kurbidaeva
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Z. M. Kachaev.

Additional information

Original Russian Text © Z.M. Kachaev, R.A. Gilmutdinov, D.V. Kopytova, A.A. Zheludkevich, Y.V. Shidlovskii, A.S. Kurbidaeva, 2017, published in Molekulyarnaya Biologiya, 2017, Vol. 51, No. 1, pp. 85–93.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kachaev, Z.M., Gilmutdinov, R.A., Kopytova, D.V. et al. RNA immunoprecipitation technique for Drosophila melanogaster S2 cells. Mol Biol 51, 72–79 (2017). https://doi.org/10.1134/S002689331606008X

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S002689331606008X

Keywords

Search

Navigation