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Experimental Validation of MicroRNA Targets Using a Luciferase Reporter System

  • Francisco E. NicolasEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 732)

Abstract

MicroRNAs (miRNAs) are a class of small noncoding transcripts that repress gene expression by pairing with their target messenger RNAs (mRNAs). The human genome codes for hundreds of different miRNAs and it is predicted that they target thousands of mRNAs involved in a wide variety of physiological processes such as development and cell identity. In animals, the identification of mRNA targets is complex because most miRNAs and their target mRNAs do not have exact or nearly exact complementarity. This tendency of animal miRNAs to bind their mRNA targets with imperfect sequence homology represents a considerable challenge to identifying miRNA targets. Computational algorithms based on conservation and experimental approaches based on expression profiles are flooding the literature with lists of candidate genes containing a large number of false-positive and false-negative predictions and indirect targets that cover the real list of direct targets for each miRNA. Currently, the only available tools to validate a sequence as a direct target of an miRNA are the systems based on a reporter gene carrying the candidate sequence. Here, an miRNA target validation reporter gene system based on the luminescence generated by the luciferase protein is described in detail, including the design of the reporter constructs, its expression in a model cell line and its measurement using a luciferase assay.

Key words

miRNA miRNA target Target validation miRNA mimics Luminescence Luciferase assay 

Notes

Acknowledgments

This work was supported by the Fundación Séneca (Murcia, Spain).

References

  1. 1.
    Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.PubMedCrossRefGoogle Scholar
  2. 2.
    Bartel, B., and Bartel, D. P. (2003). MicroRNAs: at the root of plant development? Plant Physiol 132, 709–717.PubMedCrossRefGoogle Scholar
  3. 3.
    Basyuk, E., Suavet, F., Doglio, A., Bordonne, R., and Bertrand, E. (2003). Human let-7 stem–loop precursors harbor features of RNase III cleavage products. Nucleic Acids Res 31, 6593–6597.PubMedCrossRefGoogle Scholar
  4. 4.
    Baulcombe, D. (2004). RNA silencing in plants. Nature 431, 356–363.PubMedCrossRefGoogle Scholar
  5. 5.
    Kloosterman, W. P., and Plasterk, R. H. (2006). The diverse functions of microRNAs in animal development and disease. Dev Cell 11, 441–450.PubMedCrossRefGoogle Scholar
  6. 6.
    Lee, Y., Jeon, K., Lee, J. T., Kim, S., and Kim, V. N. (2002). MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21, 4663–4670.PubMedCrossRefGoogle Scholar
  7. 7.
    Zeng, Y., and Cullen, B. R. (2003). Sequence requirements for microRNA processing and function in human cells. RNA 9, 112–123.PubMedCrossRefGoogle Scholar
  8. 8.
    Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E., and Kutay, U. (2004). Nuclear export of microRNA precursors. Science 303, 95–98.PubMedCrossRefGoogle Scholar
  9. 9.
    Yi, R., Qin, Y., Macara, I. G., and Cullen, B. R. (2003). Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17, 3011–3016.PubMedCrossRefGoogle Scholar
  10. 10.
    Grishok, A., and Sharp, P. A. (2005). Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes. Proc Natl Acad Sci U S A 102, 17360–17365.PubMedCrossRefGoogle Scholar
  11. 11.
    Hutvagner, G., and Zamore, P. D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060.PubMedCrossRefGoogle Scholar
  12. 12.
    Leuschner, P. J., Ameres, S. L., Kueng, S., and Martinez, J. (2006). Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 7, 314–320.PubMedCrossRefGoogle Scholar
  13. 13.
    Matranga, C., Tomari, Y., Shin, C., Bartel, D. P., and Zamore, P. D. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620.PubMedCrossRefGoogle Scholar
  14. 14.
    Rand, T. A., Petersen, S., Du, F., and Wang, X. (2005). Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629.PubMedCrossRefGoogle Scholar
  15. 15.
    Bagga, S., et al. (2005). Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563.PubMedCrossRefGoogle Scholar
  16. 16.
    Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008). GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat Struct Mol Biol 15, 346–353.PubMedCrossRefGoogle Scholar
  17. 17.
    Parker, J. S., Roe, S. M., and Barford, D. (2006). Molecular mechanism of target RNA transcript recognition by Argonaute-guide complexes. Cold Spring Harb Symp Quant Biol 71, 45–50.PubMedCrossRefGoogle Scholar
  18. 18.
    Petersen, C. P., Bordeleau, M. E., Pelletier, J., and Sharp, P. A. (2006). Short RNAs repress translation after initiation in mammalian cells. Mol Cell 21, 533–542.PubMedCrossRefGoogle Scholar
  19. 19.
    Pillai, R. S., et al. (2005). Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573–1576.PubMedCrossRefGoogle Scholar
  20. 20.
    Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., and Bartel, D. P. (2002). Prediction of plant microRNA targets. Cell 110, 513–520.PubMedCrossRefGoogle Scholar
  21. 21.
    Davis, E., et al. (2005). RNAi-mediated allelic trans-interaction at the imprinted Rtl1/Peg11 locus. Curr Biol 15, 743–749.PubMedCrossRefGoogle Scholar
  22. 22.
    Yekta, S., Shih, I. H., and Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596.PubMedCrossRefGoogle Scholar
  23. 23.
    Lim, L. P., Glasner, M. E., Yekta, S., Burge, C. B., and Bartel, D. P. (2003). Vertebrate microRNA genes. Science 299, 1540.PubMedCrossRefGoogle Scholar
  24. 24.
    Baek, D., Villen, J., Shin, C., Camargo, F. D., Gygi, S. P., and Bartel, D. P. (2008). The impact of microRNAs on protein output. Nature 455, 64–71.PubMedCrossRefGoogle Scholar
  25. 25.
    Sethupathy, P., Megraw, M., and Hatzigeorgiou, A. G. (2006). A guide through present computational approaches for the identification of mammalian microRNA targets. Nat Methods 3, 881–886.PubMedCrossRefGoogle Scholar
  26. 26.
    Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P., and Burge, C. B. (2003). Prediction of mammalian microRNA targets. Cell 115, 787–798.PubMedCrossRefGoogle Scholar
  27. 27.
    Lim, L. P., et al. (2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773.PubMedCrossRefGoogle Scholar
  28. 28.
    Elmen, J., et al. (2008). Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res 36, 1153–1162.PubMedCrossRefGoogle Scholar
  29. 29.
    Nicolas, F. E., et al. (2008). Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140. RNA 14, 2513–2520.PubMedCrossRefGoogle Scholar
  30. 30.
    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
  31. 31.
    Karginov, F. V., et al. (2007). A biochemical approach to identifying microRNA targets. Proc Natl Acad Sci USA 104, 19291–19296.PubMedCrossRefGoogle Scholar
  32. 32.
    Selbach, M., Schwanhausser, B., Thierfelder, N., Fang, Z., Khanin, R., and Rajewsky, N. (2008). Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63.PubMedCrossRefGoogle Scholar
  33. 33.
    Vinther, J., Hedegaard, M. M., Gardner, P. P., Andersen, J. S., and Arctander, P. (2006). Identification of miRNA targets with stable isotope labeling by amino acids in cell culture. Nucleic Acids Res 34, e107.PubMedCrossRefGoogle Scholar
  34. 34.
    Brennecke, J., Stark, A., Russell, R. B., and Cohen, S. M. (2005). Principles of microRNA-target recognition. PLoS Biol 3, e85.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.School of Biological SciencesUniversity of East AngliaNorwichUK

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