Current Genetics

, Volume 59, Issue 3, pp 139–152 | Cite as

Characterization and localization of mitochondrial DNA-encoded tRNAs and nuclear DNA-encoded tRNAs in the sea anemone Metridium senile

  • C. Timothy BeagleyEmail author
  • David R. Wolstenholme
Research Article


The mitochondrial (mt) genome of the sea anemone Metridium senile contains genes for only two transfer RNAs (tRNAs), tRNAf-Met and tRNATrp. Experiments were conducted to seek evidence for the occurrence of functional tRNAs corresponding to these genes and for the participation of nuclear DNA-encoded tRNAs in mt-protein synthesis. RNA sequences corresponding to the two mt-tRNA genes were located in mitochondria and it was shown that 3′-CC (and possibly A, but no other nucleotide) is added post-transcriptionally to the 3′ end of at least 50 % of mt-tRNAf-Met molecules and to a small fraction of the mt-tRNATrp molecules. Using specific oligonucleotide primers based on expected nuclear DNA-encoded tRNAs in a series of RACE experiments, we located the nuclear genes for tRNAGln, tRNAIle, tRNAi-Met, tRNAVal and tRNAThr. Data from Northern blot analyses indicated that mtDNA-encoded tRNAf-Met is limited to mitochondria but that nuclear DNA-encoded tRNAVal and tRNAi-Met are present in the cytoplasm and in mitochondria. These data provide direct evidence that in M. senile, mature, functional tRNAs are transcribed from the mtDNA-encoded tRNAf-Met and tRNATrp genes, and are consistent with the interpretation that both nuclear DNA-encoded tRNAVal and tRNAi-Met are utilized in mitochondrial and cytosolic protein synthesis.


Mitochondrial tRNAs Nuclear tRNAs tRNA genes CCA addition Importation 



We thank Michael Bastiani, Brenda L. Bass, Raymond F. Gesteland, David P. Goldenberg for discussions during the course of this work, and Robert Schackmann for oligonucleotides (partially subsidized by National Institutes of Health Grant CA-42014) This work was supported by National Institutes of Health Grant GM-18375 and funds from the University of Utah, and was submitted by C. T. Beagley in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biology), College of Science, University of Utah.


  1. Abascal F, Posada D, Zardoya R (2012) The evolution of the mitochondrial genetic code in arthropods revisited. Mitochondr DNA 23:84–91CrossRefGoogle Scholar
  2. Adshya S (2008) Leishmania mitochondrial tRNA importers. Int J Biochem Cell Biol 40:2681–2685CrossRefGoogle Scholar
  3. Alfonzo JD, Soll D (2009) Mitochondrial tRNA import—the challenge to understand has just begun. Biol Chem 390:717–722PubMedCrossRefGoogle Scholar
  4. Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–465PubMedCrossRefGoogle Scholar
  5. Barrell BG, Bankier AT, Drouin J (1979) A different genetic code in human mitochondria. Nature 282:189–194PubMedCrossRefGoogle Scholar
  6. Barrell BG, Anderson S, Bankier AT, de Bruijn MHL, Chen E, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG (1980) Different pattern of codon recognition by mammalian mitochondrial tRNAs. Proc Natl Acad Sci USA 77:3164–3166PubMedCrossRefGoogle Scholar
  7. Beagley CT, Macfarlane JL, Pont-Kingdon GA, Okimoto R, Okada NA, Wolstenholme DR (1995) Mitochondrial genomes of Anthozoa (Cnidaria). In: Palmieri F, Papa P, Saccone C, Gadaleta N (eds) Progress in cell research: symposium on “30 years of progress in mitochondrial bioenergetics and molecular biology”. Elsevier Science BV, Amsterdam, pp 149–153CrossRefGoogle Scholar
  8. Beagley CT, Okada NA, Wolstenholme DR (1996) Two mitochondrial group I introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase. Proc Natl Acad Sci USA 93:5619–5623PubMedCrossRefGoogle Scholar
  9. Beagley CT, Okimoto R, Wolstenholme DR (1998) The mitochondrial genome of the sea anemone, Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near standard genetic code. Genetics 148:1091–1108PubMedGoogle Scholar
  10. Beagley CT, Okimoto R, Wolstenholme DR (1999) Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm replacement loop, and a pseudo-tRNASer(UCN) gene. Genetics 152:641–652PubMedGoogle Scholar
  11. Beaton MJ, Roger AJ, Calvalier-Smith T (1998) Sequence analysis of the mitochondrial genome of Sarcophyton glaucum: conserved gene order among octocorals. J Mol Evol 47:697–708PubMedCrossRefGoogle Scholar
  12. Bernt M, Braband A, Schierwater B, Peter F. Stadler PF (2012) Genetic aspects of mitochondrial genome evolution. Mol Phylogenet Evol, in pressGoogle Scholar
  13. Bibb MH, van Etten RA, Wright CT, Walberg MW, Clayton DA (1981) Sequence and gene organization of mouse mitochondrial DNA. Cell 26:167–180PubMedCrossRefGoogle Scholar
  14. Boore JL (1999) Animal mitochondrial genomes. Nucl Acids Res 27:1767–1780PubMedCrossRefGoogle Scholar
  15. Brown TA, Cecconi C, Tkachuk AN, Bustamante C, Clayton DA (2005) Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Gene Dev 19:2466–2476PubMedCrossRefGoogle Scholar
  16. Burger G, Plante I, Lonergan KM, Gray MW (1995) The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization. J Mol Biol 245:522–537PubMedCrossRefGoogle Scholar
  17. Chomyn A, Hunkapillar MW, Attardi G (1981) Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs. Nucl Acids Res 9:867–877PubMedCrossRefGoogle Scholar
  18. Clary DO, Wolstenholme DR (1985) The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization and genetic code. J Mol Evol 22:252–271PubMedCrossRefGoogle Scholar
  19. De Bruijn MHL, Schreier PH, Eperon IC, Barrell BG, Chen EY, Armstrong PW, Wong JFH, Roe BA (1980) A mammalian mitochondrial serine transfer RNA lacking the “dihydrouridine” loop and stem. Nucl Acids Res 8:5213–5522PubMedCrossRefGoogle Scholar
  20. Deutscher MP (1990) Ribonucleases, tRNA nucleotidyltransferase, and the 3′ processing of tRNA. Prog Nucl Acid Res Mol Biol 39:209–240CrossRefGoogle Scholar
  21. Dirheimer G, Keith G, Dumas P, Westhof E (1995) Primary, secondary and tertiary structures of tRNAs. In: Soll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. ASM press, Washington DC, pp 93–126Google Scholar
  22. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) Touchdown PCR to circumvent spurious priming during gene amplification. Nucl Acids Res 19:4008PubMedCrossRefGoogle Scholar
  23. Duchene AM, Pujol C, Mare′chal-Drouard L (2009) Import of tRNAs and aminoacyl-tRNA synthetases into mitochondria. Curr Genet 55:1–18PubMedCrossRefGoogle Scholar
  24. Esseiva AC, Marechal-Drouard L, Cosset A, Schneider A (2004) The t-stem determines the cytosolic or mitochondrial localization of Trypanosomal tRNAsMet. Mol Biol Cell 15:2750–2757CrossRefGoogle Scholar
  25. Frohman MA (1990) RACE: rapid amplification of cDNA ends. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols, a guide to methods and applications. Academic Press Inc, California, pp 28–38Google Scholar
  26. Fukami H, Knowlton N (2005) Analysis of complete mitochondrial DNA sequences of three members of the coral species complex (Cnidaria, Anthozoa, Scleractinia). Coral Reefs 24:410–417CrossRefGoogle Scholar
  27. Gissi C, Iannelli F, Pesole G (2008) Evolution of the mitochondrial genome of metazoa as exemplified by comparison of congeneric species. Heredity 101:301–320PubMedCrossRefGoogle Scholar
  28. Haen KM, Pett W, Lavrov DV (2010) Parallel loss of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases and mtDNA-encoded tRNAs in Cnidaria. Mol Biol Evol 27:2216–2219PubMedCrossRefGoogle Scholar
  29. Hancock K, Hajduk SL (1990) The mitochondrial tRNAs of Trypanosoma brucei are nuclear-encoded. J Biol Chem 265:19208–19215PubMedGoogle Scholar
  30. Helfenbein KG, Fourcade HM, Vanjani RG, Boore JL (2004) The mitochondrial genome of Paraspadella gotoi is highly reduced and reveals that chaetognaths are a sister group to protostomes. Proc Natl Acad Sci USA 101:10639–10643PubMedCrossRefGoogle Scholar
  31. Helm MH, Brule F, Degoul F, Cepanec C, Leroux J-P, Geige R, Florentz C (1998) The presence of modified nucleotides is required for cloverleaf folding in a human mitochondrial tRNA. Nucl Acids Res 26:1636–1643PubMedCrossRefGoogle Scholar
  32. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J (2009) Compilation of tRNA sequences and tRNA genes. Nucleic Acids Res 37(1):D159–D162PubMedCrossRefGoogle Scholar
  33. Kayal E, Lavrov DV (2008) The mitochondrial genome of Hydra oligactis (Cnidaria, Hydrozoa) sheds new light on animal mtDNA evolution and cnidarian phylogeny. Gene 410:177–186PubMedCrossRefGoogle Scholar
  34. Lavrov DV (2007) Key transitions in animal evolution: a mitochondrial DNA perspective. Integr Comp Biol 47:734–774PubMedCrossRefGoogle Scholar
  35. Lavrov DV (2011) Key transitions in animal evolution: a mitochondrial DNA perspective. In: Desalle R, Schierwater B (eds) Key transitions in animal evolution. Science publishers & CRS Press, Enfield, pp 35–54Google Scholar
  36. Lill R, Lepier A, Schwagele F, Sprinzl M, Vogt H, Wintermeyer W (1988) Specific recognition of the 3′-terminal adenosine of tRNAPhe in the exit site of E. coli ribosomes. J Mol Biol 203:699–705CrossRefGoogle Scholar
  37. Limbach PA, Crain PF, McCloskey JA (1994) Summary: the modified nucleosides of RNA. Nucl Acids Res 22:2183–2196PubMedCrossRefGoogle Scholar
  38. Liu M, Horowitz J (1994) Functional transfer RNAs with modifications in the 3′-CCA end: differential effects on aminoacylation and polypeptide synthesis. Proc Natl Acad Sci USA 91:10389–10393PubMedCrossRefGoogle Scholar
  39. Marechal-Drouard L, Guillemaut P, Cosset A, Arbogast M, Weber F, Weil JH, Dietrich A (1990) Transfer RNAs of potato (Solanum tuberosum) mitochondria have different genetic origins. Nucl Acids Res 18:3689–3696PubMedCrossRefGoogle Scholar
  40. Marechal-Drouard L, Weil JH, Guillemaut P (1988) Import of several tRNAs from the cytoplasm into the mitochondria in bean Phaseolus vulgaris. Nucl Acids Res 16:4777–4788PubMedCrossRefGoogle Scholar
  41. Marck C, Grosjean H (2002) tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features. RNA 8:1189–1232PubMedCrossRefGoogle Scholar
  42. Martin NC (1995) Organellar tRNAs: biosynthesis and function. In: Soll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. ASM press, Washington DC, pp 127–140Google Scholar
  43. Medina M, Collins AG, Takaoka TL, Kuehl JV, Boore JL (2006) Naked corals: skeleton loss in Scleractinia. Proc Natl Acad Sci 103:9096–9100PubMedCrossRefGoogle Scholar
  44. Montiel R, Lucena MA, Medeiros J, Simoes N (2006) The complete mitochondrial genome of the entomopathogenic nematode Steinernema carpocapsae: insights into nematode mitochondrial DNA evolution and phylogeny. J Mol Evol 62:211–225PubMedCrossRefGoogle Scholar
  45. Nagaike T, Suzuki T, Tomari Y, Takemoto-Hori C, Negayama F, Watanabe K, Ueda T (2001) Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases. J Biol Chem 276:40041–40049PubMedCrossRefGoogle Scholar
  46. Okimoto R, Macfarlane JL, Wolstenholme DR (1990) A set of tRNAs that lack either the TψC arm or the dihydrouridine arm: towards a minimal tRNA adaptor. EMBO J 9:3405–3411PubMedGoogle Scholar
  47. Okimoto R, Macfarlane JL, Clary DO, Wolstenholme DR (1992) The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum. Genetics 130:471–498PubMedGoogle Scholar
  48. Pont-Kingdon GA, Beagley CT, Okimoto R, Wolstenholme DR (1994) Mitochondrial DNA of the sea anemone, Metridium senile (Cnidaria): prokaryote-like gene for tRNAf-Met and small subunit ribosomal RNA, and standard genetic code specificities for AGR and ATA codons. J Mol Evol 39:387–399PubMedCrossRefGoogle Scholar
  49. Pont-Kingdon GA, Vassort CG, Warrior R, Okimoto R, Beagley CT, Wolstenholme DR (2000) Mitochondrial DNA of Hydra attenuata (Cnidaria): a sequence that includes an end of one linear molecule and the genes for l-rRNA, tRNAf-Met, tRNATrp, COII, and ATPase8. J Mol Evol 51:404–415PubMedGoogle Scholar
  50. Pont-Kingdon GA, Okada NA, Macfarlane JL, Beagley CT, Watkins-Sims CD, Cavalier-Smith T, Clark-Walker GD, Wolstenholme DR (1998) Mitochondrial DNA of the coral Sarcophyton glaucum contains a gene for a homologue of bacterial MutS: a possible case of gene transfer from the nucleus to the mitochondrion. J Mol Evol 46:419–443PubMedCrossRefGoogle Scholar
  51. RajBhandary UL, Chow CM (1995) Initiator tRNAs and the initiation of protein synthesis. In: Soll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. ASM press, Washington DC, pp 511–528Google Scholar
  52. Randerath E, Hari P, Agrawal HP, Randerath K (1981) Rat liver mitochondrial lysine tRNA (anticodon U*UU) contains a rudimentary D-arm and 2 hypermodified nucleotides in its anticodon loop. Biochem Biophys Res Commun 103:739–744PubMedCrossRefGoogle Scholar
  53. Rickwood D, Wilson MT, Darley-Usmar VM (1987) Isolation and characteristics of intact mitochondria. In: Darley-Usmar VM, Rickwood D, Wilson MT (eds) Mitochondria: a practical approach. IRL Press, Washington DC, pp 1–16Google Scholar
  54. Roe BA, Wong JFH, Chen EY, Armstrong PW, Stankeiwicz A, Ma D-P, McDonough J (1982) Mammalian mitochondrial tRNAs: a modified nucleotide 3′ to the anticodon may modulate their codon response. In: Slonimsky P, Borst P, Attardi G (eds) Mitochondrial genes. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 45–49Google Scholar
  55. Samaha RR, Green R, Noller HF (1995) A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome. Nature (London) 377:309–314CrossRefGoogle Scholar
  56. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  57. Schneider A (2011) Mitochondrial tRNA import and its consequences for mitochondrial translation. Ann Rev Biochem 80:1033–1053PubMedCrossRefGoogle Scholar
  58. Simpson AM, Suyama Y, Dewes H, Campbell DA, Simpson L (1989) Kinetoplastid mitochondria contain functional tRNAs which are encoded in nuclear DNA and also contain small minicircle and maxicircle transcripts of unknown function. Nucl Acids Res 17:5427–5445PubMedCrossRefGoogle Scholar
  59. Shao Z, Graf S, Chaga OY, Lavrov DV (2006) Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): a linear DNA molecule encoding a putative DNA-dependant DNA polymerase. Gene 381:92–101PubMedCrossRefGoogle Scholar
  60. Sprinzl M, Horn C, Brown M, Ioudovitch A, Teinberg S (2005) Compilation of tRNA sequences and sequences of tRNA genes. Nucl Acids Res 26:148–153CrossRefGoogle Scholar
  61. Tamura K (1994) The role of the CCA sequence of tRNA in the peptidyl-transfer reaction. FEBS Lett 353:173–176PubMedCrossRefGoogle Scholar
  62. Tamura K, Nameki N, Hasegawa T, Shimizu M, Himeno H (1994) Role of the CCA terminal sequence of tRNAVal in aminoacylation with valyl-tRNA synthetase. J Biol Chem 269:22173–22177PubMedGoogle Scholar
  63. Tarassov I, Komenski P, Kolesnikova O, Martin RP, Krasheninnikov IA, Entelis N (2007) Import of nuclear DNA-encoded RNAs into mitochondria and mitochondrial translation. Cell Cycle 6(2473):2477Google Scholar
  64. Tomita K, Weiner AM (2001) Collaboration between CC- and A-adding enzymes to build and repair the 3′-terminal CCA of tRNA in Aquifex aeolicus. Science 294:1334–1336PubMedCrossRefGoogle Scholar
  65. van Oppen MJ, Catmull J, McDonald BJ, Hislop NR, Hagerman PJ, Miller DJ (2002) The mitochondrial genome of Acropora tenuis (Cnidaria; Scleractinia) contains a large group I intron and a candidate control region. J Mol Evol 55:1–13PubMedCrossRefGoogle Scholar
  66. Watanabe K, Yokobori S (2011) tRNA modification and genetic code variations in animal mitochondria. J Nucl Acids 623095Google Scholar
  67. Wolstenholme DR (1992) Animal mitochondrial DNA: structure and evolution. In: Wolstenholme DR, Jeon KW (eds) Mitochondria genomes. International review of cytology, vol 141. Academic Press, New York, pp 173–216Google Scholar
  68. Wolstenholme DR, Macfarlane JL, Okimoto R, Clary DO, Wahleithner JA (1987) Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms. Proc Natl Acad Sci USA 84:1324–1328PubMedCrossRefGoogle Scholar
  69. Wolstenholme DR, Okimoto R, Macfarlane JL (1994) Nucleotide correlations that suggest tertiary interactions in the TV-replacement loop-containing mitochondrial tRNAs of the nematodes, Caenorhabditis elegans and Ascaris suum. Nucl Acids Res 22:4300–4306PubMedCrossRefGoogle Scholar
  70. Wolstenholme DR, Fauron CM-R (1995) Mitochondrial genome organization. In Levings III CS, Kluwer VIK (eds) Advances in cellular and molecular biology of plants. Molecular biology of the mitochondria, vol 3. Academic Publishers, the Netherlands, pp 1–59Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Department of BiologyUniversity of UtahSalt Lake CityUSA

Personalised recommendations