Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable (original) (raw)

Abstract

The synthesis rates and half-lives of the individual mitochondrial ribosomal ribonucleic acid (RNA) and polyadenylic acid-containing RNA species in HeLa cells have been determined by analyzing their kinetics of labeling with [5-3H]-uridine and the changes in specific activity of the mitochondrial nucleotide precursor pools. In one experiment, a novel method for determining the nucleotide precursor pool specific activities, using nascent RNA chains, has been utilized. All mitochondrial RNA species analyzed were found to be metabolically unstable, with half-lives of 2.5 to 3.5 h for the two ribosomal RNA components and between 25 and 90 min for the various putative messenger RNAs. A cordycepin "chase" experiment yielded half-life values for the messenger RNA species which were, in general, larger by a factor of 1.5 to 2.5 than those estimated in the labeling kinetics experiments. On the basis of previous observations, a model is proposed whereby the rate of mitochondrial RNA decay is under feedback control by some mechanism linked to RNA synthesis or processing. A short half-life was determined for five large polyadenylated RNAs, which are probably precursors of mature species. A rate of synthesis of one to two molecules per minute per cell was estimated for the various H-strand-coded messenger RNA species, and a rate of synthesis 50 to 100 times higher was estimated for the ribosomal RNA species. These data indicate that the major portion of the H-strand in each mitochondrial deoxyribonucleic acid molecule is transcribed very infrequently, possibly as rarely as once or twice per cell generation. Furthermore, these results are consistent with a previously proposed model of H-strand transcription in the form of a single polycistronic molecule.

497

Images in this article

Selected References

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

  1. Aloni Y., Attardi G. Expression of the mitochondria genome in HeLa cells. IV. Titration of mitochondrial genes for 16 s, 12 s and 4 s RNA. J Mol Biol. 1971 Jan 28;55(2):271–276. doi: 10.1016/0022-2836(71)90197-5. [DOI] [PubMed] [Google Scholar]
  2. Aloni Y., Attardi G. Expression of the mitochondrial genome in HeLa cells. II. Evidence for complete transcription of mitochondrial DNA. J Mol Biol. 1971 Jan 28;55(2):251–267. doi: 10.1016/0022-2836(71)90195-1. [DOI] [PubMed] [Google Scholar]
  3. Aloni Y., Attardi G. Expression of the mitochondrial genome in HeLa cells. XI. Isolation and characterization of transcription complexes of mitochondrial DNA. J Mol Biol. 1972 Sep 28;70(2):363–373. doi: 10.1016/0022-2836(72)90545-1. [DOI] [PubMed] [Google Scholar]
  4. Aloni Y., Attardi G. Symmetrical in vivo transcription of mitochondrial DNA in HeLa cells. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1757–1761. doi: 10.1073/pnas.68.8.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Amalric F., Merkel C., Gelfand R., Attardi G. Fractionation of mitochondrial RNA from HeLa cells by high-resolution electrophoresis under strongly denaturing conditions. J Mol Biol. 1978 Jan 5;118(1):1–25. doi: 10.1016/0022-2836(78)90241-3. [DOI] [PubMed] [Google Scholar]
  6. Angerer L., Davidson N., Murphy W., Lynch D., Attardi G. An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cells mitochondrial DNA. Cell. 1976 Sep;9(1):81–90. doi: 10.1016/0092-8674(76)90054-4. [DOI] [PubMed] [Google Scholar]
  7. Attardi B., Attardi G. Expression of the mitochondrial genome in HeLa cells. I. Properties of the discrete RNA components from the mitochondrial fraction. J Mol Biol. 1971 Jan 28;55(2):231–249. doi: 10.1016/0022-2836(71)90194-x. [DOI] [PubMed] [Google Scholar]
  8. Barrell B. G., Anderson S., Bankier A. T., de Bruijn M. H., Chen E., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A. Different pattern of codon recognition by mammalian mitochondrial tRNAs. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3164–3166. doi: 10.1073/pnas.77.6.3164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Barrell B. G., Bankier A. T., Drouin J. A different genetic code in human mitochondria. Nature. 1979 Nov 8;282(5735):189–194. doi: 10.1038/282189a0. [DOI] [PubMed] [Google Scholar]
  10. Bogenhagen D., Clayton D. A. The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid. J Biol Chem. 1974 Dec 25;249(24):7991–7995. [PubMed] [Google Scholar]
  11. Cantatore P., Attardi G. Mapping of nascent light and heavy strand transcripts on the physical map of HeLa cell mitochondrial DNA. Nucleic Acids Res. 1980 Jun 25;8(12):2605–2625. doi: 10.1093/nar/8.12.2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carré D., Attardi G. Biochemical and electron microscopic characterization of DNA-RNA complexes from HeLa cell mitochondria. Biochemistry. 1978 Aug 8;17(16):3263–3273. doi: 10.1021/bi00609a014. [DOI] [PubMed] [Google Scholar]
  13. Cashel M., Lazzarini R. A., Kalbacher B. An improved method for thin-layer chromatography of nucleotide mixtures containing 32P-labelled orthophosphate. J Chromatogr. 1969 Mar 11;40(1):103–109. doi: 10.1016/s0021-9673(01)96624-5. [DOI] [PubMed] [Google Scholar]
  14. Chomyn A., Hunkapiller M. W., Attardi G. Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs. Nucleic Acids Res. 1981 Feb 25;9(4):867–877. doi: 10.1093/nar/9.4.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cowan N. J., Milstein C. Stability of cytoplasmic ribonucleic acid in a mouse myeloma: estimation of the half-life of the messenger RNA coding for an immunoglobulin light chain. J Mol Biol. 1974 Feb 5;82(4):469–481. doi: 10.1016/0022-2836(74)90242-3. [DOI] [PubMed] [Google Scholar]
  16. Crews S., Attardi G. The sequences of the small ribosomal RNA gene and the phenylalanine tRNA gene are joined end to end in human mitochondrial DNA. Cell. 1980 Mar;19(3):775–784. doi: 10.1016/s0092-8674(80)80053-5. [DOI] [PubMed] [Google Scholar]
  17. Darnell J. E., Philipson L., Wall R., Adesnik M. Polyadenylic acid sequences: role in conversion of nuclear RNA into messenger RNA. Science. 1971 Oct 29;174(4008):507–510. doi: 10.1126/science.174.4008.507. [DOI] [PubMed] [Google Scholar]
  18. Diez J., Brawerman G. Elongation of the polyadenylate segment of messenger RNA in the cytoplasm of mammalian cells. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4091–4095. doi: 10.1073/pnas.71.10.4091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Emerson C. P. Regulation of the synthesis and the stability of ribosomal RNA during contact inhibition of growth. Nat New Biol. 1971 Jul 28;232(30):101–106. doi: 10.1038/newbio232101a0. [DOI] [PubMed] [Google Scholar]
  20. Galau G. A., Lipson E. D., Britten R. J., Davidson E. H. Synthesis and turnover of polysomal mRNAs in sea urchin embryos. Cell. 1977 Mar;10(3):415–432. doi: 10.1016/0092-8674(77)90029-0. [DOI] [PubMed] [Google Scholar]
  21. Gausing K. Regulation of ribosome production in Escherichia coli: synthesis and stability of ribosomal RNA and of ribosomal protein messenger RNA at different growth rates. J Mol Biol. 1977 Sep 25;115(3):335–354. doi: 10.1016/0022-2836(77)90158-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hunt J. A. Half-life and rate of synthesis of globin messenger ribonucleic acid. Determination of half-life of messenger ribonucleic acid and its relative synthetic rate in erythroid cells. Biochem J. 1974 Mar;138(3):487–498. doi: 10.1042/bj1380487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lansman R. A., Clayton D. A. Mitochondrial protein synthesis in mouse L-cells: effect of selective nicking of mitochondrial DNA. J Mol Biol. 1975 Dec 25;99(4):777–793. doi: 10.1016/s0022-2836(75)80184-7. [DOI] [PubMed] [Google Scholar]
  25. Lynch D. C., Attardi G. Amino acid specificity of the transfer RNA species coded for by HeLa cell mitochondrial DNA. J Mol Biol. 1976 Mar 25;102(1):125–141. doi: 10.1016/0022-2836(76)90077-2. [DOI] [PubMed] [Google Scholar]
  26. Montoya J., Ojala D., Attardi G. Distinctive features of the 5'-terminal sequences of the human mitochondrial mRNAs. Nature. 1981 Apr 9;290(5806):465–470. doi: 10.1038/290465a0. [DOI] [PubMed] [Google Scholar]
  27. Murphy W. I., Attardi B., Tu C., Attardi G. Evidence for complete symmetrical transcription in vivo of mitochondrial DNA in HeLa cells. J Mol Biol. 1975 Dec 25;99(4):809–814. doi: 10.1016/s0022-2836(75)80187-2. [DOI] [PubMed] [Google Scholar]
  28. Murphy W., Attardi G. Stability of cytoplasmic messenger RNA in HeLa cels. Proc Natl Acad Sci U S A. 1973 Jan;70(1):115–119. doi: 10.1073/pnas.70.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Neuhard J., Randerath E., Randerath K. Ion-exchange thin-layer chromatography. 13. Resolution of complex nucleoside triphosphate mixtures. Anal Biochem. 1965 Nov;13(2):211–222. doi: 10.1016/0003-2697(65)90191-0. [DOI] [PubMed] [Google Scholar]
  30. Nobrega F. G., Tzagoloff A. Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B. J Biol Chem. 1980 Oct 25;255(20):9828–9837. [PubMed] [Google Scholar]
  31. Ojala D. K., Montoya J., Attardi G. The putative mRNA for subunit II of human cytochrome c oxidase starts directly at the translation initiator codon. Nature. 1980 Sep 4;287(5777):79–82. doi: 10.1038/287079a0. [DOI] [PubMed] [Google Scholar]
  32. Ojala D., Attardi G. Expression of the mitochondrial genome in HeLa cells. X. Properties of mitochondrial polysomes. J Mol Biol. 1972 Mar 28;65(2):273–289. doi: 10.1016/0022-2836(72)90282-3. [DOI] [PubMed] [Google Scholar]
  33. Ojala D., Attardi G. Fine mapping of the ribosomal RNA genes of HeLa cell mitochondrial DNA. J Mol Biol. 1980 Apr;138(2):411–420. doi: 10.1016/0022-2836(80)90296-x. [DOI] [PubMed] [Google Scholar]
  34. Ojala D., Attardi G. Identification and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by HeLa cell mitochondrial DNA. J Mol Biol. 1974 Sep 5;88(1):205–219. doi: 10.1016/0022-2836(74)90305-2. [DOI] [PubMed] [Google Scholar]
  35. Ojala D., Merkel C., Gelfand R., Attardi G. The tRNA genes punctuate the reading of genetic information in human mitochondrial DNA. Cell. 1980 Nov;22(2 Pt 2):393–403. doi: 10.1016/0092-8674(80)90350-5. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Palmiter R. D. Rate of ovalbumin messenger ribonucleic acid synthesis in the oviduct of estrogen-primed chicks. J Biol Chem. 1973 Dec 10;248(23):8260–8270. [PubMed] [Google Scholar]
  38. Penman S., Rosbash M., Penman M. Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1878–1885. doi: 10.1073/pnas.67.4.1878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Perry R. P., Kelley D. E. Messenger RNA turnover in mouse L cells. J Mol Biol. 1973 Oct 5;79(4):681–696. doi: 10.1016/0022-2836(73)90071-5. [DOI] [PubMed] [Google Scholar]
  40. Posakony J. W., England J. M., Attardi G. Mitochondrial growth and division during the cell cycle in HeLa cells. J Cell Biol. 1977 Aug;74(2):468–491. doi: 10.1083/jcb.74.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Singer R. H., Penman S. Messenger RNA in HeLa cells: kinetics of formation and decay. J Mol Biol. 1973 Aug 5;78(2):321–334. doi: 10.1016/0022-2836(73)90119-8. [DOI] [PubMed] [Google Scholar]
  42. Thalenfeld B. E., Tzagoloff A. Assembly of the mitochondrial membrane system. Sequence of the oxi 2 gene of yeast mitochondrial DNA. J Biol Chem. 1980 Jul 10;255(13):6173–6180. [PubMed] [Google Scholar]
  43. Zylber E. A., Perlman S., Penman S. Mitochondrial RNA turnover in the presence of cordycepin. Biochim Biophys Acta. 1971 Jul 29;240(4):588–593. doi: 10.1016/0005-2787(71)90716-7. [DOI] [PubMed] [Google Scholar]
  44. Zylber E., Vesco C., Penman S. Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide. J Mol Biol. 1969 Aug 28;44(1):195–204. doi: 10.1016/0022-2836(69)90414-8. [DOI] [PubMed] [Google Scholar]