Abstract
As we trek into the uncharted territories of the genomic era, there is an urgency for the development of approaches for assigning functions to the multitude of uncharacterized genes. Although currently available knock-out methodologies could be used for uncovering the function of newly discovered genes, the mixed outcomes in terms of the success of these approaches in down-regulating gene expression necessitate the development of new functional genomics tools. This chapter describes in detail the experimental method for targeted mRNA degradation inside plant cells by enticing the endogenous and ubiquitous RNase P into recognition of specific mRNAs as non-natural substrates.
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References
Martienssen, R. A. (1998) Functional genomics: probing plant gene function and expression with transposons. Proc. Natl. Acad. Sci. USA 95, 2021–2026.
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.
van der Kol, A. R., Lenting, P. E., Veenstra, J., et al. (1988) An antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333, 866–869.
Tanner, N. K. (1999) Ribozymes: the characteristics and properties of catalytic RNAs. FEMS Microbiol. Rev. 23, 257–275.
Storz, G. (2002) An expanding universe of noncoding RNAs. Science 296, 1260–1262.
Merlo, A. O., Cowen, N., Delate, T., et al. (1998) Ribozymes targeted to stearoyl-ACP Δ9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves. Plant Cell 10, 1603–1621.
Chuang, C.-F. and Meyerowitz, E. M. (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis. Proc. Natl. Acad. Sci. USA 97, 4985–4990.
Gopalan, V., Vioque, A., and Altman, S. (2002) RNase P: variations and uses. J. Biol. Chem. 277, 6759–6762.
Hall, T. A. and Brown, J. W. (2001) The ribonuclease P family. Methods Enzymol. 341, 56–77.
Xiao, S., Scott, F., Fierke, C. A., and Engelke, D. R. (2002) Eukaryotic ribonuclease P: a plurality of ribonucleoprotein enzymes. Annu. Rev. Biochem. 71, 165–189.
Forster, A. C. and Altman, S. (1990) External guide sequence for an RNA enzyme. Science 249, 783–786.
Guerrier-Takada, C. and Altman, S. (2000) Inactivation of gene expression using ribonuclease P and external guide sequences. Methods Enzymol. 313, 442–456.
Plehn-Dujowich, D. and Altman, S. (1998) Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P. Proc. Natl. Acad. Sci. USA 95, 7327–7332.
Yen, L., Gonzalez-Zulueta, M., Feldman, A., et al. (2001) Reduction of functional N-methyl-D-aspartate receptors in neurons by RNase P-mediated cleavage of the NR1 mRNA. J. Neurochem. 76,1386–1394.
Dunn, W., Trang, P., Khan, U., Zhu, J., and Liu, F. (2001) RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences. Proc. Natl. Acad. Sci. USA 98, 14831–14836.
Ma, M., Benimetskaya, L., Lebedeva, I., Dignam, J., Takle, G., and Stein, C. A. (2000) Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. Nat. Biotechnol. 18, 58–61.
Cobaleda, C. and Sachez-Garcia, I. (2001) RNase P: from biological function to biotechnological applications. Trends Biotechnol. 19, 406–411.
Raj, M. L. S., Pulukkunat, D. K., Reckard, J. F., Thomas, G., and Gopalan, V. (2001) Cleavage of bipartite substrates by rice and maize ribonuclease P. Application to degradation of target mRNAs in plants. Plant Physiol. 125, 1187–1190.
Zuker, M. and Stiegler, P. (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 9, 133–148.
Moine, H., Ehresmann, B., Ehresmann, C., and Romby, P. (1997) Probing RNA structure and function in solution, in RNA Structure and Function (Simons, R. W. and Grunberg-Manago, M., eds.), CSH Laboratory Press, Cold Spring Harbor, NY, pp. 77–116.
Allawi, H. T., Dong, F., Ip, H. S., Neri, B. P., and Lyamichev, V. I. (2001) Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase. RNA 7, 314–327.
Pan, W., Devlin, H. F., Kelly, C., Isom, H. C., and Clawson, G. A. (2001) A selection system for identifying accessible sites in target RNAs. RNA 7, 610–620.
Amarzguioui, M., Brede, G., Babaie, E., Grotli, M., Sproat, B., and Prydz, H. (2000) Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes. Nucleic Acids Res. 28, 4113–4124.
Stiffler, M. A. (2002) Substrate recognition by Zea mays RNase P: implications for an RNase P-based functional genomics approach in plants. B.S. (Honors) Thesis, The Ohio State University, Columbus, OH.
Yuan, Y. and Altman, S. (1995) Substrate recognition by human RNase P: identification of small, model substrates for the enzyme. EMBO J. 14, 159–168.
Waibel, F. and Filipowicz, W. (1990) U6 snRNA genes of Arabidopsis are transcribed by RNA polymerase III but contain the same two upstream promoter elements as RNA polymerase II-transcribed U-snRNA genes. Nucleic Acids Res. 18, 3451–3458.
Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions. Nucleic Acids Res. 18, 3459–3466.
Heard, D. J., Filipowicz, W., Marques, J. P., Palme, K., and Gualberto, J. M. (1995) An upstream U-SnRNA gene-like promoter is required for the transcription of the Arabidopsis thaliana 7SL RNA gene. Nucleic Acids Res. 23, 1970.
Connelly, S. and Filipowicz, W. (1993) Activity of chimeric U snRNA/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U SnRNA 3′-end formation and transcription initiation can occur independently in plants. Mol. Cell. Biol. 13, 6403–6415.
Connelly, S., Marshallsay, C., Leader, D., Brown, J. W., and Filipowicz, W. (1994) Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts. Mol. Cell. Biol. 14, 5910–5919.
Bertrand, E., Houser-Scott, F., Kendall, A., Singer, R. H., and Engelke, D. R. (1998) Nucleolar localization of early tRNA processing. Genes Dev. 12, 2463–2468.
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Tsai, H.-Y., Lai, L. B., and Gopalan, V. (2002) A modified pBluescript-based vector for facile cloning and transcription of RNAs. Anal. Biochem. 303, 214–217.
Vioque, A., Arnez, J., and Altman, S. (1988) Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli. J. Mol. Biol. 202, 835–848.
Hartmann, R. K., Krupp, G., and Hardt, W.-D. (1995) Towards a new concept of gene inactivation: specific RNA cleavage by endogenous RNase P. Annu. Rev. Biotechnol. 1, 215–265.
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Pulukkunat, D.K., Raj, M.L.S., Pattanayak, D., Lai, L.B., Gopalan, V. (2003). Exploring the Potential of Plant RNase P as a Functional Genomics Tool. In: Grotewold, E. (eds) Plant Functional Genomics. Methods in Molecular Biology™, vol 236. Humana Press. https://doi.org/10.1385/1-59259-413-1:295
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DOI: https://doi.org/10.1385/1-59259-413-1:295
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