Identifying Specific Trans-Factors of RNA Editing in Plant Mitochondria by Multiplex Single Base Extension Typing

  • Mizuki TakenakaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 718)


The multiplex single base extension SNP-typing procedure outlined here can be employed to screen large numbers of plants for mutations in nuclear genes that affect mitochondrial RNA editing. The high ­sensitivity of this method allows high-throughput analysis of individual plants altered in RNA editing at given sites in total cellular cDNA from pooled RNA preparations of up to 50 green plants. The method can be used for large-scale screening for mutations in genes encoding trans-factors for specific RNA ­editing sites. Several nuclear encoded genes involved in RNA editing at specific sites in mitochondria of Arabidopsis thaliana have been identified by this approach.

Key words

RNA editing Plant mitochondria Mutant screening SNP typing 



I thank Dagmar Pruchner for excellent experimental help. I am very grateful to Walther Vogel, Christiane Maier and Bärbel Weber in the Institut für Humangenetik, Universität Ulm, for the use of the automated sequencing equipment and materials and their very constructive and helpful discussions throughout. This work was supported by grants from the DFG.


  1. 1.
    Giegé, P., and Brennicke, A. (1999) RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs. Proc Natl Acad Sci U S A 96, 15324–15329.PubMedCrossRefGoogle Scholar
  2. 2.
    Takenaka, M., Verbitskiy, D., van der Merwe, J. A., Zehrmann, A., and Brennicke, A. (2008) The process of RNA editing in plant mitochondria. Mitochondrion 8, 35–46.PubMedCrossRefGoogle Scholar
  3. 3.
    Farré, J.-C., Leon, G., Jordana, X., and Araya, A. (2001) cis Recognition elements in plant mitochondrion RNA editing. Mol Cell Biol 21, 6731–6737.PubMedCrossRefGoogle Scholar
  4. 4.
    Neuwirt, J., Takenaka, M., van der Merwe, J. A., and Brennicke, A. (2005) An in vitro RNA editing system from cauliflower mitochondria: editing site recognition parameters can vary in different plant species. RNA 11, 1563–1570.PubMedCrossRefGoogle Scholar
  5. 5.
    van der Merwe, J. A., Takenaka, M., Neuwirt, J., Verbitskiy, D., and Brennicke, A. (2006) RNA editing sites in plant mitochondria can share cis-elements. FEBS Lett 580, 268–272.PubMedCrossRefGoogle Scholar
  6. 6.
    Zehrmann, A., Verbitskiy, D., van der Merwe, J. A., Brennicke, A., and Takenaka, M. (2009) A DYW-domain containing PPR-protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell 21, 558–567.Google Scholar
  7. 7.
    Zehrmann, A., van der Merwe, J. A., Verbitskiy, D., Brennicke, A., and Takenaka, M. (2008) Ecotype-specific variations in the extent of RNA editing in plant mitochondria. Mitochondrion 8, 319–327.PubMedCrossRefGoogle Scholar
  8. 8.
    Kotera, E., Tasaka, M., and Shikanai, T. (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433, 326–330.PubMedCrossRefGoogle Scholar
  9. 9.
    Okuda, K., Myouga, R., Motohashi, K., Shinozaki, K., and Shikanai, T. (2007) Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc Natl Acad Sci U S A 104, 8178–8183.CrossRefGoogle Scholar
  10. 10.
    Okuda, K., Nakamura, T., Sugita, M., Shimizu, T., and Shikanai, T. (2006) A pentatricopeptide repeat protein is a site-recognition factor in chloroplast RNA editing. J Biol Chem 281, 37661–37667.PubMedCrossRefGoogle Scholar
  11. 11.
    Chateigner-Boutin, A.-L., Ramos-Vega, M., Guevara-Garcia, A., Andrés, C., Gutierrez-Nava, M. d. l. L., Cantero, A., Delannoy, E., Jimenez, L. F., Lurin, C., Small, I. D., and León, P. (2008) CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts. Plant J 56, 590–602.PubMedCrossRefGoogle Scholar
  12. 12.
    Takenaka, M., and Brennicke, A. (2008) Multiplex single base extension typing to identify nuclear genes required for RNA editing in plant organelles. Nucleic Acids Res. doi:  10.1093/nar/gkn975 PubMedGoogle Scholar
  13. 13.
    Lindblad-Toh, K., Winchester, E., Daly, M. J., Wang, D. G., Hirschhorn, J. N., Laviolette, J.-P., Ardlie, K., Reich, D. E., Robinson, E., Sklar, P., Shah, N., Thomas, D., Fan, J. B., Gingeras, T., Warrington, J., Patil, N., Hudson, T. J., and Lander, E. S. (2000) Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nat Genet 24, 381–386.PubMedCrossRefGoogle Scholar
  14. 14.
    Nelson, T. M., Just, R. S., Loreille, O., Schanfield, M. S., and Podini, S. (2007) Development of a multiplex single base extension assay for mitochondrial DNA haplotype typing. Croat Med J 48, 460–472.PubMedGoogle Scholar
  15. 15.
    Sasaki, T., Yukawa, Y., Wakasugi, T., Yamada, K., and Sugiura, M. (2006) A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts. Plant J 47, 802–810.PubMedCrossRefGoogle Scholar
  16. 16.
    Chateigner-Boutin, A.-L., and Small, I. D. (2007) A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons. Nucleic Acids Res 35, e114.PubMedCrossRefGoogle Scholar
  17. 17.
    Lurin, C., Andrés, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyère, C., Caboche, M., Debast, C., Gualberto, J. M., Hoffmann, B., Lecharny, A., Le Ret, M., Martin-Magniette, M.-L., Mireau, H., Peeters, N., Renou, J.-P., Szurek, B., Taconnat, L., and Small, I. D. (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103.PubMedCrossRefGoogle Scholar
  18. 18.
    Andrés, C., Lurin, C., and Small, I. D. (2007) The multifarious roles of PPR proteins in plant mitochondrial gene expression. Physiol Plant 129, 14–22.CrossRefGoogle Scholar
  19. 19.
    Takenaka, M., and Brennicke, A. (2007) RNA editing in plant mitochondria: assays and biochemical approaches. Methods Enzymol 424, 439–458.PubMedGoogle Scholar
  20. 20.
    Arabidopsis Protocols (Martínez-Zapater, J. M., and Salinas, J. eds.), (1998) Methods in Molecular Biology 82, Humana, Totowa, NJ.Google Scholar
  21. 21.
    Plant Gene Transfer and Expression Protocols (Jones, H. ed.), (1995) Methods in Molecular Biology 49, Humana, Totowa, NJ.Google Scholar
  22. 22.
    Picardi, E., Regina, T. M. R., Brennicke, A., and Quagliariello, C. (2007) REDIdb: the RNA editing database. Nucleic Acids Res 35, D173–D177.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Molekulare BotanikUniversität UlmUlmGermany

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