Methods for Analysis of Mitochondrial tRNA Editing in Acanthamoeba castellanii

Part of the Methods in Molecular Biology book series (MIMB, volume 265)


We present here methodology for assaying 5′-terminal editing of mitochondrial tRNAs in the amoeboid protist Acanthamoeba castellanii. This type of editing involves replacement of one or more nucleotides within the first three positions at the 5′ end of a tRNA substrate. The assay procedure involves RNA ligase-mediated joining of the 5′ and 3′ ends of a tRNA, use of the resulting circularized tRNA as template for cDNA synthesis primed by tRNA-specific primers over a region that encompasses the ligated 5′ and 3′ halves of the acceptor stem, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of double-stranded cDNA product. This approach has the advantage that it simultaneously reveals potential editing events on the 5′ and 3′ side of an acceptor stem, as well as serves to identify mature tRNAs (characterized by having a 3′-CCAOH motif) and partially processed intermediates.

Key Words

Acanthamoeba castellanii mitochondria mitochondrial isolation RNA ligase reverse transcription polymerase chain reaction tRNA circularization tRNA editing 


  1. 1.
    Price, D. H. and Gray, M. W. (1999) Confirmation of predicted edits and demonstration of unpredicted edits in Acanthamoeba castellanii mitochondrial tRNAs. Curr. Genet. 35, 23–29.PubMedCrossRefGoogle Scholar
  2. 2.
    Price, D. H. and Gray, M. W. (1998) Editing of tRNA, in Modification and Editing of RNA (Grosjean, H. and Benne, R., eds.), ASM Press, Washington, DC, pp. 289–305.Google Scholar
  3. 3.
    Gray, M. W. (2001) Speculations on the origin and evolution of RNA editing, in RNA Editing (Bass, B. L., ed.), Oxford University Press, Oxford, UK, pp. 160–184.Google Scholar
  4. 4.
    Hopper, A. K. and Phizicky, E. M. (2003) tRNA transfers to the limelight. Genes Dev. 17, 162–180.PubMedCrossRefGoogle Scholar
  5. 5.
    Fey, J., Weil, J. H., Tomita, K., Cosset, A., Dietrich, A., Small, I., and Maréchal-Drouard, L. (2002) Role of editing in plant mitochondrial transfer RNAs. Gene 286, 21–24.PubMedCrossRefGoogle Scholar
  6. 6.
    Janke, A. and Pääbo, S. (1993) Editing of a tRNA anticodon in marsupial mitochondria changes its codon recognition. Nucleic Acids Res. 21, 1523–1525.PubMedCrossRefGoogle Scholar
  7. 7.
    Miller, D., Mahendran, R., Spottswood, M., Costandy, H., Wang, S., Ling, M.-L., and Yang, N. (1993) Insertional editing in mitochondria of Physarum. Semin. Cell Biol. 4, 261–266.PubMedCrossRefGoogle Scholar
  8. 8.
    Antes, T., Costandy, H., Mahendran, R., Spottswood, M., and Miller, D. (1998) Insertional editing of mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes. Mol. Cell. Biol. 18, 7521–7527.PubMedGoogle Scholar
  9. 9.
    Yokobori, S.-I. and Pääbo, S. (1995) tRNA editing in metazoans. Nature 377, 490.PubMedCrossRefGoogle Scholar
  10. 10.
    Yokobori, S.-I. and Pääbo, S. (1995) Transfer RNA editing in land snail mitochondria. Proc. Natl Acad. Sci. USA 92, 10,432–10,435.PubMedCrossRefGoogle Scholar
  11. 11.
    Tomita, K., Ueda, T., and Watanabe, K. (1996) RNA editing in the acceptor stem of squid mitochondrial tRNATyr. Nucleic Acids Res. 24, 4987–4991.PubMedCrossRefGoogle Scholar
  12. 12.
    Yokobori, S.-I. and Pääbo, S. (1997) Polyadenylation creates the discriminator nucleotide of chicken mitochondrial tRNATyr. J. Mol. Biol. 265, 95–99.PubMedCrossRefGoogle Scholar
  13. 13.
    Hatzoglou, E., Rodakis, G. C., and Lecanidou, R. (1995) Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea. Genetics 140, 1353–1366.PubMedGoogle Scholar
  14. 14.
    Yamazaki, N., Ueshima, R., Terrett, J. A., et al. (1997) Evolution of pulmonate gastropod mitochondrial genomes: comparisons of gene organizations of Euhadra, Cepaea and Albinaria and implications of unusual tRNA secondary structures. Genetics 145, 749–758.PubMedGoogle Scholar
  15. 15.
    Lonergan, K. M. and Gray, M. W. (1993) Editing of transfer RNAs in Acanthamoeba castellanii. Science 259, 812–816.PubMedCrossRefGoogle Scholar
  16. 16.
    Lonergan, K. M. and Gray, M. W. (1993) Predicted editing of additional transfer RNAs in Acanthamoeba castellanii mitochondria. Nucleic Acids Res. 21, 4402.PubMedCrossRefGoogle Scholar
  17. 17.
    Cole, R. A. and Williams, K. L. (1994) The Dictyostelium discoideum mitochondrial genome: a primordial system using the universal code and encoding hydrophilic proteins atypical of metazoan mitochondrial DNA. J. Mol. Evol. 39, 579–588.PubMedCrossRefGoogle Scholar
  18. 18.
    Angata, K., Kuroe, K., Yanagisawa, K., and Tanaka, Y. (1995) Codon usage, genetic code and phylogeny of Dictyostelium discoideum mitochondrial DNA as deduced from a 7.3-kb region. Curr. Genet. 27, 249–256.PubMedCrossRefGoogle Scholar
  19. 19.
    Laforest, M.-J., Roewer, I., and Lang, B. F. (1997) Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG “stop” codons recognized as leucine. Nucleic Acids Res. 25, 626–632.PubMedCrossRefGoogle Scholar
  20. 20.
    Forget, L., Ustinova, J., Wang, Z., Huss, V. A. R., and Lang, B. F. (2002) Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi. Mol. Biol. Evol. 19, 310–319.PubMedGoogle Scholar
  21. 21.
    Lavrov, D. V., Brown, W. M., and Boore, J. L. (2000) A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus. Proc. Natl. Acad. Sci. USA 97, 13,738–13,742.PubMedCrossRefGoogle Scholar
  22. 22.
    Burger, G., Plante, I., Lonergan, K. M., and Gray, M. W. (1995) The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization. J. Mol. Biol. 245, 522–537.PubMedCrossRefGoogle Scholar
  23. 23.
    Price, D. H. and Gray, M. W. (1999) A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii. RNA 5, 302–317.PubMedCrossRefGoogle Scholar
  24. 24.
    Sugino, A., Snopek, T. J., and Cozzarelli, N. R. (1977) Bacteriophage T4 RNA ligase: reaction intermediates and interaction of substrates. J. Biol. Chem. 252, 1732–1737.PubMedGoogle Scholar
  25. 25.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  26. 26.
    Spencer, D. F., Gray, M. W., and Schnare, M. N. (1992) The isolation of wheat mitochondrial DNA and RNA, in Seed Analysis, vol. 14 in Modern Methods of Plant Analysis, New Series (Linskens, H. F. and Jackson, J. F., eds.), Springer-Verlag, Berlin, pp. 347–360.Google Scholar
  27. 27.
    Parish, J. H. and Kirby, K. S. (1966) Reagents which reduce interactions between ribosomal RNA and rapidly labelled RNA from rat liver. Biochim. Biophys. Acta 129, 554–562.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyDalhousie UniversityHalifaxCanada

Personalised recommendations