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 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
de Carvalho, F., Gheysen, G., Kushnir, S., Van Montagu, M., Inze, D., and Castresana, C. (1992) Suppression of b-1,3-glucanase transgene expression in homozygous plants. EMBO J. 11, 2595–2602.
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.
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.
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.
Chapman, E. J. and Carrington, J. C. (2007) Specialization and evolution of endogenous small RNA pathways. Nat. Rev. Genet. 8, 884–896.
Ossowski, S., Schwab, R., and Weigel, D. (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J. 53, 674–90.
Ghildiyal, M. and Zamore, P. D. (2009) Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94–108.
Ambros, V. (2003) MicroRNA pathways in flies and worms: Growth, death, fat, stress, and timing. Cell 113, 673–676.
Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297.
Cai, X., Hagedorn, C. H., and Cullen, B. R. (2004) Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10, 1957–1966.
Kim, V. N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Cell Biol. 6, 376–385.
Xie, Z., Johansen, L. K., Gustafson, A. M., Kasschau, K. D., Lellis, A. D., Zilberman, D., Jacobsen, S. E., and Carrington, J. C. (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, E104.
Han, M. H., Goud, S., Song, L., and Fedoroff, N. (2004) The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl. Acad. Sci. USA 101, 1093–1098.
Fang, Y. and Spector, D. L. (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr. Biol. 17, 818–23.
Kurihara, Y., Takashi, Y., and Watanabe, Y. (2006) The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12, 206–12.
Song, L., Axtell, M. J., and Fedoroff, N. V. RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr. Biol. 20, 37–41.
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.
Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., and Poethig, R. S. (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl. Acad. Sci. USA 102, 3691–3696.
Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R. W., Steward, R., and Chen, X. (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935.
Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J., Hammond, S. M., Joshua-Tor, L., and Hannon, G. J. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.
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.
Khraiwesh, B., Arif, M. A., Seumel, G. I., Ossowski, S., Weigel, D., Reski, R., and Frank, W. Transcriptional control of gene expression by microRNAs. Cell 140, 111–122.
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.
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.
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.
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.
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.
Cho, S. H., Addo-Quaye, C., Coruh, C., Arif, M. A., Ma, Z., Frank, W., and Axtell, M. J. (2008) Physcomitrella patens DCL3 is required for 22–24 nt siRNA accumulation, suppression of retrotransposon-derived transcripts, and normal development. PLoS Genet. 4, e1000314.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Kim, Y. S., Ke, F., Lei, X. Y., Zhu, R., and Zhang, Q. Y. (2010) Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish cells by AmiRNA. PLoS One. 5, e10308.
Zhang, J., Liu, Q. S., and Dong, W. G. (2011) Blockade of proliferation and migration of gastric cancer via targeting CDH17 with an artificial microRNA. Med Oncol. 28, 494–501.
De Guire, V., Caron, M., Scott, N., Menard, C., Gaumont-Leclerc, M. F., Chartrand, P., Major, F., and Ferbeyre, G. (2010) Designing small multiple-target artificial RNAs. Nucleic Acids Res. 38, e140.
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.
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.
Qu, J., Ye, J., and Fang, R. (2007) Artificial microRNA-mediated virus resistance in plants. J. Virol. 81, 6690–6699.
Fernandez, A. I., Viron, N., Alhagdow, M., Karimi, M., Jones, M., Amsellem, Z., Sicard, A., Czerednik, A., Angenent, G., Grierson, D., May, S., Seymour, G., Eshed, Y., Lemaire-Chamley, M., Rothan, C., and Hilson, P. (2009) Flexible tools for gene expression and silencing in tomato. Plant Physiol. 151, 1729–1740.
Wang, X., Yang, Y., Yu, C., Zhou, J., Cheng, Y., Yan, C., and Chen, J. (2010) A highly efficient method for construction of rice artificial microRNA vectors. Mol. Biotechnol. 46, 211–218
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.
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.
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.
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.
Frank, W., Decker, E. L., and Reski, R. (2005) Molecular tools to study Physcomitrella patens. Plant Biol. 7, 220–227.
Volloch, V., Schweitzer, B., and Rits, S. (1994) Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of b globin mRNA with an extended 5’-untranslated region. Nucleic Acids Res. 22, 2507–2511.
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.
Acknowledgments
This work was supported by Landesstiftung Baden-Württemberg (P-LS-RNS/40 to W.F.) and the German Academic Exchange Service (DAAD; PhD fellowships to I.F. and M.A.A.).
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Fattash, I., Khraiwesh, B., Arif, M.A., Frank, W. (2012). Expression of Artificial MicroRNAs in Physcomitrella patens . In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_25
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DOI: https://doi.org/10.1007/978-1-61779-558-9_25
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