Abstract
The mammalian mitochondrial genome contains 37 genes, 13 of which encode polypeptide subunits in the enzyme complexes of the oxidative phosphorylation system. The other genes encode the rRNAs and tRNAs necessary for their translation. The mitochondrial translation machinery is located in the mitochondrial matrix, and is exclusively dedicated to the synthesis of these 13 enzyme subunits. Mitochondrial disease in humans is often associated with defects in mitochondrial translation. This can manifest as a global decrease in the rate of mitochondrial protein synthesis, a decrease in the synthesis of specific polypeptides, the synthesis of abnormal polypeptides, or in altered stability of specific translation products. All of these changes in the normal pattern of mitochondrial translation can be assessed by a straightforward technique that takes advantage of the insensitivity of the mitochondrial translation machinery to antibiotics that completely inhibit cytoplasmic translation. Thus, specific radioactive labeling of the mitochondrial translation products can be achieved in cultured cells, and the results can be visualized on gradient gels. The analysis of mitochondrial translation in cells cultured from patient biopsies is useful in the study of disease-causing mutations in both the mitochondrial and the nuclear genomes.
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References
Chomyn, A. (1996) In vivo labeling and analysis of human mitochondrial translation products. Methods Enzymol, 264, 197–211.
Fernandez-Silva, P., Acin-Perez, R., Fernandez-Vizarra, E., Perez-Martos, A. and Enriquez, J.A. (2007) In vivo and in organello analyses of mitochondrial translation. Methods Cell Biol, 80, 571–588.
Costantino, P. and Attardi, G. (1977) Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells. J Biol Chem, 252, 1702–1711.
Mariottini, P., Chomyn, A., Doolittle, R.F. and Attardi, G. (1986) Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex. J Biol Chem, 261, 3355–3362.
Chomyn, A., Martinuzzi, A., Yoneda, M., Daga, A., Hurko, O., Johns, D., Lai, S.T., Nonaka, I., Angelini, C. and Attardi, G. (1992) MELAS mutation in mtDNA binding site for transcription termination factor causes defects in protein synthesis and in respiration but no change in levels of upstream and downstream mature transcripts. Proc Natl Acad Sci USA, 89, 4221–4225.
Chomyn, A., Meola, G., Bresolin, N., Lai, S.T., Scarlato, G. and Attardi, G. (1991) In vitro genetic transfer of protein synthesis and respiration defects to mitochondrial DNA-less cells with myopathy-patient mitochondria. Mol Cell Biol, 11, 2236–2244.
Enriquez, J.A., Chomyn, A. and Attardi, G. (1995) MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet, 10, 47–55.
Hayashi, J., Ohta, S., Kikuchi, A., Takemitsu, M., Goto, Y. and Nonaka, I. (1991) Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction. Proc Natl Acad Sci USA, 88, 10614–10618.
King, M.P., Koga, Y., Davidson, M. and Schon, E.A. (1992) Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNA(Leu(UUR)) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. Mol Cell Biol, 12, 480–490.
Sasarman, F., Antonicka, H. and Shoubridge, E.A. (2008) The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chain assembly defect partially suppressed by overexpression of EFTu and EFG2. Hum Mol Genet, 17, 3697–3707.
Antonicka, H., Ostergaard, E., Sasarman, F., Weraarpachai, W., Wibrand, F., Pedersen, A.M., Rodenburg, R.J., van der Knaap, M.S., Smeitink, J.A., Chrzanowska-Lightowlers, Z.M. et al. (2010) Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. Am J Hum Genet, 87, 115–122.
Antonicka, H., Sasarman, F., Kennaway, N.G. and Shoubridge, E.A. (2006) The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. Hum Mol Genet, 15, 1835–1846.
Coenen, M.J., Antonicka, H., Ugalde, C., Sasarman, F., Rossi, R., Heister, J.G., Newbold, R.F., Trijbels, F.J., van den Heuvel, L.P., Shoubridge, E.A. et al. (2004) Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med, 351, 2080–2086.
Fernandez-Vizarra, E., Berardinelli, A., Valente, L., Tiranti, V. and Zeviani, M. (2007) Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA). J Med Genet, 44, 173–180.
Kemp, J.P., Smith, P.M., Pyle, A., Neeve, V.C., Tuppen, H.A., Schara, U., Talim, B., Topaloglu, H., Holinski-Feder, E., Abicht, A. et al. (2011) Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency. Brain, 134, 183–195.
Miller, C., Saada, A., Shaul, N., Shabtai, N., Ben-Shalom, E., Shaag, A., Hershkovitz, E. and Elpeleg, O. (2004) Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation. Ann Neurol, 56, 734–738.
Riley, L.G., Cooper, S., Hickey, P., Rudinger-Thirion, J., McKenzie, M., Compton, A., Lim, S.C., Thorburn, D., Ryan, M.T., Giege, R. et al. (2010) Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia – MLASA syndrome. Am J Hum Genet, 87, 52–59.
Smeitink, J.A., Elpeleg, O., Antonicka, H., Diepstra, H., Saada, A., Smits, P., Sasarman, F., Vriend, G., Jacob-Hirsch, J., Shaag, A. et al. (2006) Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs. Am J Hum Genet, 79, 869–877.
Valente, L., Tiranti, V., Marsano, R.M., Malfatti, E., Fernandez-Vizarra, E., Donnini, C., Mereghetti, P., De Gioia, L., Burlina, A., Castellan, C. et al. (2007) Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. Am J Hum Genet, 80, 44–58.
Zeharia, A., Shaag, A., Pappo, O., Mager-Heckel, A.M., Saada, A., Beinat, M., Karicheva, O., Mandel, H., Ofek, N., Segel, R. et al. (2009) Acute infantile liver failure due to mutations in the TRMU gene. Am J Hum Genet, 85, 401–407.
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Sasarman, F., Shoubridge, E.A. (2012). Radioactive Labeling of Mitochondrial Translation Products in Cultured Cells. In: Wong, Ph.D., LJ. (eds) Mitochondrial Disorders. Methods in Molecular Biology, vol 837. Humana Press. https://doi.org/10.1007/978-1-61779-504-6_14
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DOI: https://doi.org/10.1007/978-1-61779-504-6_14
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