RNAi pp 29-43 | Cite as

In vitro RNA Cleavage Assay for Argonaute-Family Proteins

  • Keita Miyoshi
  • Hiroshi Uejima
  • Tomoko Nagami-Okada
  • Haruhiko Siomi
  • Mikiko C. Siomi
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 442)

Summary

Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage (“Slicing”) of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage (“Slicer”) activity of the RISC. We describe (in vitro) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli.

Key Words

RNAi Argonaute Slicer RISC Drosophila 
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Notes

Acknowledgments

K. M. is a research fellow supported by the Japan Society for the Promotion of Science (JSPS). This work was supported by grants to M. C. S. and H. S. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the JSPS, and the New Energy and Industrial Technology Development Organization. M.C.S. is also supported by CREST from Japan Science and Technology Agency.

References

  1. 1.
    Tomari, Y., and Zamore, P. D. (2005). Perspective: Machines for RNAi. Genes Dev. 19, 517–529.CrossRefPubMedGoogle Scholar
  2. 2.
    Sontheimer, E. J. (2005). Assembly and function of RNA silencing complexes. Nat. Rev. Mol. Cell Biol. 6, 127–138.CrossRefPubMedGoogle Scholar
  3. 3.
    Bernstein, E., Caudy, A. A., Hammond, S. M., and Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366.CrossRefPubMedGoogle Scholar
  4. 4.
    Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200.CrossRefPubMedGoogle Scholar
  5. 5.
    Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J., and Plasterk, R. H. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659.CrossRefPubMedGoogle Scholar
  6. 6.
    Knight, S. W., and Bass, B. L. (2001). A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 2932269–2271.CrossRefPubMedGoogle Scholar
  7. 7.
    Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R., and Hannon, G. J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296.CrossRefPubMedGoogle Scholar
  8. 8.
    Hutvagner, G., and Zamore, P. D. (2002). RNAi: Nature abhors a double-strand. Curr. Opin. Genet. Dev. 12, 225–232.CrossRefPubMedGoogle Scholar
  9. 9.
    Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574.CrossRefPubMedGoogle Scholar
  10. 10.
    Nykanen, A., Haley, B., and Zamore, P. D. (2001). ATP requirements and small interfering RNA structure in the RNA interference pathway.Cell 107, 309–321.CrossRefPubMedGoogle Scholar
  11. 11.
    Hutvagner, G., and Zamore, P. D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060.CrossRefPubMedGoogle Scholar
  12. 12.
    Ambros, W. (2004). The functions of animal microRNAs. Nature 431, 350–355.CrossRefPubMedGoogle Scholar
  13. 13.
    Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116, 281–297.CrossRefPubMedGoogle Scholar
  14. 14.
    Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.CrossRefPubMedGoogle Scholar
  15. 15.
    Olsen, P. H., and Ambros, V. (1999). The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev. Biol. 216 671–680.CrossRefPubMedGoogle Scholar
  16. 16.
    Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R., and Ruvkun, G. (2000). The 21-nucleotide hlet-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906.CrossRefPubMedGoogle Scholar
  17. 17.
    Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B., and Cohen, S. M. (2003). Bantam encodes a developmentally regulated microRNA that controls cell proli-feration and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36.CrossRefPubMedGoogle Scholar
  18. 18.
    Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294, 853–858.CrossRefPubMedGoogle Scholar
  19. 19.
    Lau, N. C., Lim, L. P., Weinstein, E. G., and Bartel, D. P. (2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294 858–862.CrossRefPubMedGoogle Scholar
  20. 20.
    Lee, R. C., and Ambros, V. (2001). An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864.CrossRefPubMedGoogle Scholar
  21. 21.
    Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and Bartel, D. P. (2002). MicroRNAs in plants. Genes Dev. 16, 1616–1629.CrossRefPubMedGoogle Scholar
  22. 22.
    Song, J.-J., Smith, S. K., Hannon, G. J., and Joshua-Tor, L. (2004). Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437.CrossRefPubMedGoogle Scholar
  23. 23.
    Liu, J., Carmell, M. A., Rivas, F. V., et al. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.CrossRefPubMedGoogle Scholar
  24. 24.
    Rivas, F. V., Tolia, N. H., Song, J.-J., et al. (2005). Purified Argonaute2 and an siRNA form recombinant human RISC. Nat. Struct. Mol. Biol. 12, 340–349.CrossRefPubMedGoogle Scholar
  25. 25.
    Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and Tuschl, T. (2004). Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197.CrossRefPubMedGoogle Scholar
  26. 26.
    Sasaki, T., Shiohama, A., Minoshima, S., and Shimizu, N. (2003).Identification of eight members of the Argonaute family in the human genome. Genomics 82, 323–330.CrossRefPubMedGoogle Scholar
  27. 27.
    Williams, R. W., and Rubin, G. M. (2002). ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos. Proc. Natl. Acad. Sci. USA 99, 6889–6894.Google Scholar
  28. 28.
    Okamura, K., Ishizuka, A., Siomi, H., and Siomi, M. C. (2004). Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655–1666.CrossRefPubMedGoogle Scholar
  29. 29.
    Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H., and Siomi, M. C. (2005). Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19, 2837–2848.CrossRefPubMedGoogle Scholar
  30. 30.
    Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000). RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33.CrossRefPubMedGoogle Scholar
  31. 31.
    Tomari, Y., Matranga, C., Haley, B., Martinez, N., and Zamore P. D. (2004). A protein sensor for siRNA asymmetry. Science 306, 1377–1380.CrossRefPubMedGoogle Scholar
  32. 32.
    Kiriakidou, M., Nelson, P., Lamprinaki, S., Sharma, A., and Mourelatos, Z. (2005). Detection of microRNAs and assays to monitor microRNA activities in vivo and in vitro. Meth. Mol. Biol. 309, 295–310.Google Scholar
  33. 33.
    Tolla, N. H., and Joshua-Tor, L. (2006). Strategies for protein coexpression in Escherichia coli. Nat. Meth. 3, 55–64.CrossRefGoogle Scholar
  34. 34.
    Dorner, S., Lum, L., Kim, M., Paro, R., Beachy, P. A., and Green, R. (2006). A genomewide screen for components of the RNAi pathway in Proc. Natl. Acad. Sci. USA 103, 11880–11885.Google Scholar
  35. 35.
    Hall, T. M. T. (2005). Structure and function of Argonaute proteins. Structure 13, 1403–1408.CrossRefPubMedGoogle Scholar
  36. 36.
    Martinez, J., and Tuschl, T. (2004). RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Keita Miyoshi
    • 1
  • Hiroshi Uejima
    • 1
  • Tomoko Nagami-Okada
    • 1
  • Haruhiko Siomi
    • 1
  • Mikiko C. Siomi
    • 2
  1. 1.Institute for Genome Research, University of TokushimaTokushimaJapan
  2. 2.Institute for Genome Research, University of Tokushima JST, CRESTTokushimaJapan

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