Functional polypeptides can be synthesized from human mitochondrial transcripts lacking termination codons (original) (raw)

Abstract

The human mitochondrial genome (mtDNA) is a small, circular DNA duplex found in multi-copy in the mitochondrial matrix. It is almost fully transcribed from both strands to produce large polycistronic RNA units that are processed and matured. The 13 mtDNA-encoded polypeptides are translated from mt-mRNAs that have been matured by polyadenylation of their free 3'-termini. A patient with clinical features consistent with an mtDNA disorder was recently shown to carry a microdeletion, resulting in the loss of the termination codon for MTATP6 and in its juxtaposition with MTCO3. Cell lines from this patient exhibited low steady-state levels of RNA14, the bi-cistronic transcript encoding subunits 6 and 8 of the F(o)F(1)-ATP synthase, complex V, consistent with a decreased stability. Recent reports of 'non-stop' mRNA decay systems in the cytosol have failed to determine the fate of gene products derived from transcripts lacking termination codons, although enhanced decay clearly required the 'non-stop' transcripts to be translated. We wished to determine whether functional translation products could still be expressed from non-stop transcripts in the human mitochondrion. Although a minor defect in complex V assembly was noted in the patient-derived cell lines, the steady-state level of ATPase 6 was similar to controls, consistent with the pattern of de novo mitochondrial protein synthesis. Moreover, no significant difference in ATP synthase activity could be detected. We conclude that, in the absence of a functional termination codon, although mitochondrial transcripts are more rapidly degraded, they are also translated to generate stable polypeptides that are successfully integrated into functional enzyme complexes.

Full Text

The Full Text of this article is available as a PDF (185.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson S., Bankier A. T., Barrell B. G., de Bruijn M. H., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A., Sanger F. Sequence and organization of the human mitochondrial genome. Nature. 1981 Apr 9;290(5806):457–465. doi: 10.1038/290457a0. [DOI] [PubMed] [Google Scholar]
  2. Andrews R. M., Kubacka I., Chinnery P. F., Lightowlers R. N., Turnbull D. M., Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet. 1999 Oct;23(2):147–147. doi: 10.1038/13779. [DOI] [PubMed] [Google Scholar]
  3. Cai Y. C., Bullard J. M., Thompson N. L., Spremulli L. L. Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts. J Biol Chem. 2000 Jul 7;275(27):20308–20314. doi: 10.1074/jbc.M001899200. [DOI] [PubMed] [Google Scholar]
  4. Chinnery P. F., Johnson M. A., Wardell T. M., Singh-Kler R., Hayes C., Brown D. T., Taylor R. W., Bindoff L. A., Turnbull D. M. The epidemiology of pathogenic mitochondrial DNA mutations. Ann Neurol. 2000 Aug;48(2):188–193. [PubMed] [Google Scholar]
  5. Chinnery P. F., Turnbull D. M. Epidemiology and treatment of mitochondrial disorders. Am J Med Genet. 2001 Spring;106(1):94–101. doi: 10.1002/ajmg.1426. [DOI] [PubMed] [Google Scholar]
  6. Chomyn A. In vivo labeling and analysis of human mitochondrial translation products. Methods Enzymol. 1996;264:197–211. doi: 10.1016/s0076-6879(96)64020-8. [DOI] [PubMed] [Google Scholar]
  7. DiMauro Salvatore, Schon Eric A. Mitochondrial respiratory-chain diseases. N Engl J Med. 2003 Jun 26;348(26):2656–2668. doi: 10.1056/NEJMra022567. [DOI] [PubMed] [Google Scholar]
  8. Frischmeyer Pamela A., van Hoof Ambro, O'Donnell Kathryn, Guerrerio Anthony L., Parker Roy, Dietz Harry C. An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science. 2002 Mar 22;295(5563):2258–2261. doi: 10.1126/science.1067338. [DOI] [PubMed] [Google Scholar]
  9. Frolova L. Y., Tsivkovskii R. Y., Sivolobova G. F., Oparina N. Y., Serpinsky O. I., Blinov V. M., Tatkov S. I., Kisselev L. L. Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA. 1999 Aug;5(8):1014–1020. doi: 10.1017/s135583829999043x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Herrnstadt Corinna, Elson Joanna L., Fahy Eoin, Preston Gwen, Turnbull Douglass M., Anderson Christen, Ghosh Soumitra S., Olefsky Jerrold M., Beal M. Flint, Davis Robert E. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. Am J Hum Genet. 2002 Apr 5;70(5):1152–1171. doi: 10.1086/339933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirsch M., Penman S. Post-transcriptional addition of polyadenylic acid to mitochondrial RNA by a cordycepin-insensitive process. J Mol Biol. 1974 Feb 25;83(2):131–142. doi: 10.1016/0022-2836(74)90384-2. [DOI] [PubMed] [Google Scholar]
  12. James A. M., Sheard P. W., Wei Y. H., Murphy M. P. Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations. Eur J Biochem. 1999 Jan;259(1-2):462–469. doi: 10.1046/j.1432-1327.1999.00066.x. [DOI] [PubMed] [Google Scholar]
  13. Keiler K. C., Waller P. R., Sauer R. T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science. 1996 Feb 16;271(5251):990–993. doi: 10.1126/science.271.5251.990. [DOI] [PubMed] [Google Scholar]
  14. Kisselev Lev, Ehrenberg Måns, Frolova Ludmila. Termination of translation: interplay of mRNA, rRNAs and release factors? EMBO J. 2003 Jan 15;22(2):175–182. doi: 10.1093/emboj/cdg017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maquat L. E., Carmichael G. G. Quality control of mRNA function. Cell. 2001 Jan 26;104(2):173–176. doi: 10.1016/s0092-8674(01)00202-1. [DOI] [PubMed] [Google Scholar]
  16. Menzies Fiona M., Cookson Mark R., Taylor Robert W., Turnbull Douglass M., Chrzanowska-Lightowlers Zofia M. A., Dong Lichun, Figlewicz Denise A., Shaw Pamela J. Mitochondrial dysfunction in a cell culture model of familial amyotrophic lateral sclerosis. Brain. 2002 Jul;125(Pt 7):1522–1533. doi: 10.1093/brain/awf167. [DOI] [PubMed] [Google Scholar]
  17. Muto A., Ushida C., Himeno H. A bacterial RNA that functions as both a tRNA and an mRNA. Trends Biochem Sci. 1998 Jan;23(1):25–29. doi: 10.1016/s0968-0004(97)01159-6. [DOI] [PubMed] [Google Scholar]
  18. Nagaike T., Suzuki T., Tomari Y., Takemoto-Hori C., Negayama F., Watanabe K., Ueda T. Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases. J Biol Chem. 2001 Aug 14;276(43):40041–40049. doi: 10.1074/jbc.M106202200. [DOI] [PubMed] [Google Scholar]
  19. Nakamura Yoshikazu, Ito Koichi. Making sense of mimic in translation termination. Trends Biochem Sci. 2003 Feb;28(2):99–105. doi: 10.1016/S0968-0004(03)00006-9. [DOI] [PubMed] [Google Scholar]
  20. Nijtmans Leo G. J., Henderson Nadine S., Holt Ian J. Blue Native electrophoresis to study mitochondrial and other protein complexes. Methods. 2002 Apr;26(4):327–334. doi: 10.1016/S1046-2023(02)00038-5. [DOI] [PubMed] [Google Scholar]
  21. Ojala D., Montoya J., Attardi G. tRNA punctuation model of RNA processing in human mitochondria. Nature. 1981 Apr 9;290(5806):470–474. doi: 10.1038/290470a0. [DOI] [PubMed] [Google Scholar]
  22. Oldfors Anders, Tulinius Már. Mitochondrial encephalomyopathies. J Neuropathol Exp Neurol. 2003 Mar;62(3):217–227. doi: 10.1093/jnen/62.3.217. [DOI] [PubMed] [Google Scholar]
  23. Piwowarski Jan, Grzechnik Pawel, Dziembowski Andrzej, Dmochowska Aleksandra, Minczuk Michal, Stepien Piotr P. Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria. J Mol Biol. 2003 Jun 20;329(5):853–857. doi: 10.1016/s0022-2836(03)00528-x. [DOI] [PubMed] [Google Scholar]
  24. Puranam R. S., Attardi G. The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P. Mol Cell Biol. 2001 Jan;21(2):548–561. doi: 10.1128/MCB.21.2.548-561.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schägger H., von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991 Dec;199(2):223–231. doi: 10.1016/0003-2697(91)90094-a. [DOI] [PubMed] [Google Scholar]
  26. Seit-Nebi A., Frolova L., Justesen J., Kisselev L. Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition. Nucleic Acids Res. 2001 Oct 1;29(19):3982–3987. doi: 10.1093/nar/29.19.3982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seneca S., Abramowicz M., Lissens W., Muller M. F., Vamos E., de Meirleir L. A mitochondrial DNA microdeletion in a newborn girl with transient lactic acidosis. J Inherit Metab Dis. 1996;19(2):115–118. doi: 10.1007/BF01799407. [DOI] [PubMed] [Google Scholar]
  28. Temperley Richard J., Seneca Sara H., Tonska Katarzyna, Bartnik Ewa, Bindoff Laurence A., Lightowlers Robert N., Chrzanowska-Lightowlers Zofia M. A. Investigation of a pathogenic mtDNA microdeletion reveals a translation-dependent deadenylation decay pathway in human mitochondria. Hum Mol Genet. 2003 Jul 22;12(18):2341–2348. doi: 10.1093/hmg/ddg238. [DOI] [PubMed] [Google Scholar]
  29. Wanders R. J., Ruiter J. P., Wijburg F. A. Studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts: application to mitochondrial encephalomyopathies. Biochim Biophys Acta. 1993 Jun 19;1181(3):219–222. doi: 10.1016/0925-4439(93)90024-u. [DOI] [PubMed] [Google Scholar]
  30. Zhang Y., Spremulli L. L. Identification and cloning of human mitochondrial translational release factor 1 and the ribosome recycling factor. Biochim Biophys Acta. 1998 Nov 26;1443(1-2):245–250. doi: 10.1016/s0167-4781(98)00223-1. [DOI] [PubMed] [Google Scholar]
  31. van Hoof Ambro, Frischmeyer Pamela A., Dietz Harry C., Parker Roy. Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science. 2002 Mar 22;295(5563):2262–2264. doi: 10.1126/science.1067272. [DOI] [PubMed] [Google Scholar]