Experimental Identification of MicroRNA Targets by Immunoprecipitation of Argonaute Protein Complexes

  • Michaela Beitzinger
  • Gunter MeisterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 732)


MicroRNAs (miRNAs) represent a class of small noncoding RNAs that negatively regulate gene expression­. Intensive research during the past decade has established miRNAs as key regulators of many cellular pathways. MiRNAs have also been implicated in a number of diseases including various forms of cancer. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and function in multi-protein complexes. MiRNAs guide Ago protein complexes to partially complementary sequences typically located in the 3′ untranslated region (UTR) of their target mRNAs leading to the inhibition of its translation and/or its destabilization. To understand the biological roles of miRNAs, it is essential to identify the mRNA targets that they regulate. Because of the low degree of complementarity between the miRNA and its target sequence, it is often difficult to find targets computationally. Therefore, biochemical methods are needed to identify miRNA targets experimentally. The availability of highly specific monoclonal antibodies against Argonaute proteins allows for the isolation of functional Ago-miRNA–mRNA complexes from ­different cell lines, tissues, or even patient samples. Here we provide a detailed protocol for isolation and identification of miRNA target mRNAs from immunoprecipitated human Ago protein complexes.

Key words

Argonaute proteins MicroRNAs Immunoprecipitation Gene silencing mRNAs Dicer Drosha 



We thank Sabine Rottmüller and Bernd Haas for technical ­assistance, Elisabeth Kremmer for antibody production, and Vladimir Benes for affymetrix array analysis. This work was in part supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, FO855), the European Union (LSHG-CT-2006-037900), and the Max-Planck-Society.


  1. 1.
    Bartel, D. P. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136, 215–33.PubMedCrossRefGoogle Scholar
  2. 2.
    Carthew, R. W., and Sontheimer, E. J. (2009)Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642–55.Google Scholar
  3. 3.
    Chen, P. Y., and Meister, G. (2005) microRNA-guided posttranscriptional gene regulation. Biol Chem 386, 1205–18.PubMedCrossRefGoogle Scholar
  4. 4.
    Meister, G., and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Parker, J. S., and Barford, D. (2006) Argonaute: A scaffold for the function of short regulatory RNAs. Trends Biochem Sci 31, 622–30.PubMedCrossRefGoogle Scholar
  6. 6.
    Peters, L., and Meister, G. (2007) Argonaute proteins: mediators of RNA silencing. Mol Cell 26, 611–23.PubMedCrossRefGoogle Scholar
  7. 7.
    Tolia, N. H., and Joshua-Tor, L. (2007) Slicer and the argonautes. Nat Chem Biol 3, 36–43.PubMedCrossRefGoogle Scholar
  8. 8.
    Jinek, M., and Doudna, J. A. (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–12.PubMedCrossRefGoogle Scholar
  9. 9.
    Chen, K., and Rajewsky, N. (2007) The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet 8, 93–103.PubMedCrossRefGoogle Scholar
  10. 10.
    Beitzinger, M., Peters, L., Zhu, J. Y., Kremmer, E., and Meister, G. (2007) Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol 4, 76–84.PubMedCrossRefGoogle Scholar
  11. 11.
    Easow, G., Teleman, A. A., and Cohen, S. M. (2007) Isolation of microRNA targets by miRNP immunopurification. RNA 13, 1198–204.PubMedCrossRefGoogle Scholar
  12. 12.
    Hendrickson, D. G., Hogan, D. J., Herschlag, D., Ferrell, J. E., and Brown, P. O. (2008) Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance. PLoS ONE 3, e2126.PubMedCrossRefGoogle Scholar
  13. 13.
    Karginov, F. V., Conaco, C., Xuan, Z., Schmidt, B. H., Parker, J. S., Mandel, G., and Hannon, G. J. (2007) A biochemical approach to identifying microRNA targets. Proc Natl Acad Sci U S A 104, 19291–6.PubMedCrossRefGoogle Scholar
  14. 14.
    Landthaler, M., Gaidatzis, D., Rothballer, A., Chen, P. Y., Soll, S. J., Dinic, L., Ojo, T., Hafner, M., Zavolan, M., and Tuschl, T. (2008) Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–96.PubMedCrossRefGoogle Scholar
  15. 15.
    Weinmann, L., Hock, J., Ivacevic, T., Ohrt, T., Mutze, J., Schwille, P., Kremmer, E., Benes, V., Urlaub, H., and Meister, G. (2009) Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs. Cell 136, 496–507.PubMedCrossRefGoogle Scholar
  16. 16.
    Rudel, S., Flatley, A., Weinmann, L., Kremmer, E., and Meister, G. (2008) A multifunctional human Argonaute2-specific monoclonal antibody. RNA 14, 1244–53.PubMedCrossRefGoogle Scholar
  17. 17.
    Pall, G. S., and Hamilton, A. J. (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3, 1077–84.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.Laboratory for RNA Biology, Center for integrated protein science Munich (CIPSM)Max-Planck-Institute of BiochemistryMartinsriedGermany

Personalised recommendations