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Gene Function Analysis by Artificial MicroRNAs in Physcomitrella patens

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RNAi and Plant Gene Function Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 744))

Abstract

MicroRNAs (miRNAs) are ~21 nt long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis amiRNAs can be designed to target any gene of interest. During the last decade the moss Physcomitrella patens emerged as a model plant for functional gene analysis based on its unique ability to integrate DNA into the nuclear genome by homologous recombination which allows for the generation of targeted gene knockout mutants. In addition to this, we developed a protocol to express amiRNAs in P. patens that has particular advantages over the generation of knockout mutants and might be used to speed up reverse genetics approaches in this model species.

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References

  1. de Carvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., Inze, D., and Castresana, C. (1992) Suppression of β-1,3-glucanase transgene expression in homozygous plants. EMBO J. 11, 2595–2602.

    PubMed  Google Scholar 

  2. 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.

    Article  PubMed  CAS  Google Scholar 

  3. Matzke, M. A., Primig, M., Trnovsky, J., and Matzke, A. J. (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8, 643–649.

    PubMed  CAS  Google Scholar 

  4. Napoli, C., Lemieux, C., and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279–289.

    Article  PubMed  CAS  Google Scholar 

  5. Tomari, Y. and Zamore, P. D. (2005) Perspective: machines for RNAi. Genes Dev. 19, 517–529.

    Article  PubMed  CAS  Google Scholar 

  6. Hamilton, A. J. and Baulcombe, D. C. (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952.

    Article  PubMed  CAS  Google Scholar 

  7. Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.

    Article  PubMed  CAS  Google Scholar 

  8. Chapman, E. J. and Carrington, J. C. (2007) Specialization and evolution of endogenous small RNA pathways. Nat. Rev. Genet. 8, 884–896.

    Article  PubMed  CAS  Google Scholar 

  9. Kurihara, Y. and Watanabe, Y. (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. USA 101, 12753–12758.

    Article  PubMed  CAS  Google Scholar 

  10. Jones-Rhoades, M. W., Bartel, D. P., and Bartel, B. (2006) MicroRNAs and their regulatory roles in plants. Annu. Rev. Plant. Biol. 57, 19–53.

    Article  PubMed  CAS  Google Scholar 

  11. Axtell, M. J., Snyder, J. A., and Bartel, D. P. (2007) Common functions for diverse small RNAs of land plants. Plant Cell 19, 1750–1769.

    Article  PubMed  CAS  Google Scholar 

  12. Fattash, I., Voss, B., Reski, R., Hess, W. R., and Frank, W. (2007) Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution. BMC Plant Biol. 7, 13.

    Article  PubMed  Google Scholar 

  13. Aravin, A. A., Lagos-Quintana, M., Yalcin, A., Zavolan, M., Marks, D., Snyder, B., Gaasterland, T., Meyer, J., and Tuschl, T. (2003) The small RNA profile during Drosophila melanogaster development. Dev. Cell 5, 337–350.

    Article  PubMed  CAS  Google Scholar 

  14. Ossowski, S., Schwab, R., and Weigel, D. (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J. 53, 674–690.

    Article  PubMed  CAS  Google Scholar 

  15. Allen, E., Xie, Z., Gustafson, A. M., and Carrington, J. C. (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221.

    Article  PubMed  CAS  Google Scholar 

  16. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L., and Poethig, R. S. (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379.

    Article  PubMed  CAS  Google Scholar 

  17. Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A. C., Hilbert, J. L., Bartel, D. P., and Crete, P. (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79.

    Article  PubMed  CAS  Google Scholar 

  18. Yoshikawa, M., Peragine, A., Park, M. Y., and Poethig, R. S. (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 2164–2175.

    Article  PubMed  CAS  Google Scholar 

  19. Axtell, M. J., Jan, C., Rajagopalan, R., and Bartel, D. P. (2006) A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565–577.

    Article  PubMed  CAS  Google Scholar 

  20. Fahlgren, N., Montgomery, T. A., Howell, M. D., Allen, E., Dvorak, S. K., Alexander, A. L., and Carrington, J. C. (2006) Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr. Biol. 16, 939–944.

    Article  PubMed  CAS  Google Scholar 

  21. Hunter, C., Willmann, M. R., Wu, G., Yoshikawa, M., de la Luz Gutierrez-Nava, M., and Poethig, S. R. (2006) Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis. Development 133, 2973–2981.

    Article  PubMed  CAS  Google Scholar 

  22. Guo, H. S., Xie, Q., Fei, J. F., and Chua, N. H. (2005) MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17, 1376–1386.

    Article  PubMed  CAS  Google Scholar 

  23. Vaucheret, H., Vazquez, F., Crete, P., and Bartel, D. P. (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18, 1187–1197.

    Article  PubMed  CAS  Google Scholar 

  24. Alvarez, J. P., Pekker, I., Goldshmidt, A., Blum, E., Amsellem, Z., and Eshed, Y. (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18, 1134–1151.

    Article  PubMed  CAS  Google Scholar 

  25. Niu, Q. W., Lin, S. S., Reyes, J. L., Chen, K. C., Wu, H. W., Yeh, S. D., and Chua, N. H. (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat. Biotechnol. 24, 1420–1428.

    Article  PubMed  CAS  Google Scholar 

  26. Parizotto, E. A., Dunoyer, P., Rahm, N., Himber, C., and Voinnet, O. (2004) In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev. 18, 2237–2242.

    Article  PubMed  CAS  Google Scholar 

  27. Schwab, R., Ossowski, S., Riester, M., Warthmann, N., and Weigel, D. (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18, 1121–1133.

    Article  PubMed  CAS  Google Scholar 

  28. Warthmann, N., Chen, H., Ossowski, S., Weigel, D., and Herve, P. (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS ONE 3, e1829.

    Article  PubMed  Google Scholar 

  29. Zeng, Y., Wagner, E. J., and Cullen, B. R. (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333.

    Article  PubMed  CAS  Google Scholar 

  30. Boden, D., Pusch, O., Silbermann, R., Lee, F., Tucker, L., and Ramratnam, B. (2004) Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res. 32, 1154–1158.

    Article  PubMed  CAS  Google Scholar 

  31. Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J., and Lowe, S. W. (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet. 37, 1289–1295.

    PubMed  CAS  Google Scholar 

  32. Qu, J., Ye, J., and Fang, R. (2007) Artificial microRNA-mediated virus resistance in plants. J. Virol. 81, 6690–6699.

    Article  PubMed  CAS  Google Scholar 

  33. Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., and Frank, W. (2008) Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Plant Physiol. 148, 684–693.

    Article  PubMed  CAS  Google Scholar 

  34. Schwab, R., Palatnik, J. F., Riester, M., Schommer, C., Schmid, M., and Weigel, D. (2005) Specific effects of microRNAs on the plant transcriptome. Dev. Cell 8, 517–527.

    Article  PubMed  CAS  Google Scholar 

  35. Mallory, A. C., Reinhart, B. J., Jones-Rhoades, M. W., Tang, G., Zamore, P. D., Barton, M. K., and Bartel, D. P. (2004) MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ region. EMBO J. 23, 3356–3364.

    Article  PubMed  CAS  Google Scholar 

  36. Koncz, C., Martini, N., Mayerhofer, R., Koncz-Kalman, Z., Korber, H., Redei, G. P., and Schell, J. (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc. Natl. Acad. Sci. USA 86, 8467–8471.

    Article  PubMed  CAS  Google Scholar 

  37. Frank, W., Decker, E. L., and Reski, R. (2005) Molecular tools to study Physcomitrella patens. Plant Biol. 7, 220–227.

    Article  PubMed  CAS  Google Scholar 

  38. Volloch, V., Schweitzer, B., and Rits, S. (1994) Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of β globin mRNA with an extended 5′-untranslated region. Nucleic Acids Res. 22, 2507–2511.

    Article  PubMed  CAS  Google Scholar 

  39. Llave, C., Xie, Z., Kasschau, K. D., and Carrington, J. C. (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053–2056.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We gratefully acknowledge financial support from the German Academic Exchange Service (DAAD; Ph.D. fellowships to I.F. and M.A.A.).

Author information

Authors and Affiliations

  1. Center for Plant Stress Genomics and Technology, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia

    Basel Khraiwesh

  2. Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent University, Ghent, Belgium

    Basel Khraiwesh

  3. Department of Plant Biotechnology and Genetics, Ghent University, Ghent, Belgium

    Basel Khraiwesh

  4. Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestraße 1, 79104, Freiburg, Germany

    Isam Fattash, M. Asif Arif & Wolfgang Frank

Authors
  1. Basel Khraiwesh
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  2. Isam Fattash
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  3. M. Asif Arif
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  4. Wolfgang Frank
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Corresponding author

Correspondence to Basel Khraiwesh .

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Editors and Affiliations

  1. Graduate School of Horticulture, Chiba University, Yayoi-cho 1-33, Chiba, 263-8522, Japan

    Hiroaki Kodama

  2. Evolutionary Biology, Research Institute of, Kamiyoga 2-4-28, Tokyo, Setagaya, 158-0098, Japan

    Atsushi Komamine

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Khraiwesh, B., Fattash, I., Arif, M.A., Frank, W. (2011). Gene Function Analysis by Artificial MicroRNAs in Physcomitrella patens . In: Kodama, H., Komamine, A. (eds) RNAi and Plant Gene Function Analysis. Methods in Molecular Biology, vol 744. Humana Press. https://doi.org/10.1007/978-1-61779-123-9_5

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  • DOI: https://doi.org/10.1007/978-1-61779-123-9_5

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-122-2

  • Online ISBN: 978-1-61779-123-9

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