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. 1984 Jul;3(7):1567–1572. doi: 10.1002/j.1460-2075.1984.tb02012.x

Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.

C Jacq, J Banroques, A M Becam, P P Slonimski, N Guiso, A Danchin
PMCID: PMC557560  PMID: 6430696

Abstract

Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.

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Selected References

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  1. Anziano P. Q., Hanson D. K., Mahler H. R., Perlman P. S. Functional domains in introns: trans-acting and cis-acting regions of intron 4 of the cob gene. Cell. 1982 Oct;30(3):925–932. doi: 10.1016/0092-8674(82)90297-5. [DOI] [PubMed] [Google Scholar]
  2. Bonitz S. G., Berlani R., Coruzzi G., Li M., Macino G., Nobrega F. G., Nobrega M. P., Thalenfeld B. E., Tzagoloff A. Codon recognition rules in yeast mitochondria. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3167–3170. doi: 10.1073/pnas.77.6.3167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonitz S. G., Coruzzi G., Thalenfeld B. E., Tzagoloff A., Macino G. Assembly of the mitochondrial membrane system. Structure and nucleotide sequence of the gene coding for subunit 1 of yeast cytochrme oxidase. J Biol Chem. 1980 Dec 25;255(24):11927–11941. [PubMed] [Google Scholar]
  4. Burke J. M., RajBhandary U. L. Intron within the large rRNA gene of N. crassa mitochondria: a long open reading frame and a consensus sequence possibly important in splicing. Cell. 1982 Dec;31(3 Pt 2):509–520. doi: 10.1016/0092-8674(82)90307-5. [DOI] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Carignani G., Groudinsky O., Frezza D., Schiavon E., Bergantino E., Slonimski P. P. An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae. Cell. 1983 Dec;35(3 Pt 2):733–742. doi: 10.1016/0092-8674(83)90106-x. [DOI] [PubMed] [Google Scholar]
  7. Cech T. R., Tanner N. K., Tinoco I., Jr, Weir B. R., Zuker M., Perlman P. S. Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3903–3907. doi: 10.1073/pnas.80.13.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Church G. M., Slonimski P. P., Gilbert W. Pleiotropic mutations within two yeast mitochondrial cytochrome genes block mRNA processing. Cell. 1979 Dec;18(4):1209–1215. doi: 10.1016/0092-8674(79)90233-2. [DOI] [PubMed] [Google Scholar]
  9. Côté C., Solioz M., Schatz G. Biogenesis of the cytochrome bc1 complex of yeast mitochondria. A precursor form of the cytoplasmically made subunit V. J Biol Chem. 1979 Mar 10;254(5):1437–1439. [PubMed] [Google Scholar]
  10. Davies R. W., Waring R. B., Ray J. A., Brown T. A., Scazzocchio C. Making ends meet: a model for RNA splicing in fungal mitochondria. Nature. 1982 Dec 23;300(5894):719–724. doi: 10.1038/300719a0. [DOI] [PubMed] [Google Scholar]
  11. De La Salle H., Jacq C., Slonimski P. P. Critical sequences within mitochondrial introns: pleiotropic mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase. Cell. 1982 Apr;28(4):721–732. doi: 10.1016/0092-8674(82)90051-4. [DOI] [PubMed] [Google Scholar]
  12. Dhawale S., Hanson D. K., Alexander N. J., Perlman P. S., Mahler H. R. Regulatory interactions between mitochondrial genes: interactions between two mosaic genes. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1778–1782. doi: 10.1073/pnas.78.3.1778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Douglas M. G., Butow R. A. Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1083–1086. doi: 10.1073/pnas.73.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dujardin G., Jacq C., Slonimski P. P. Single base substitution in an intron of oxidase gene compensates splicing defects of the cytochrome b gene. Nature. 1982 Aug 12;298(5875):628–632. doi: 10.1038/298628a0. [DOI] [PubMed] [Google Scholar]
  15. Grossman A. D., Burgess R. R., Walter W., Gross C. A. Mutations in the Ion gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase. Cell. 1983 Jan;32(1):151–159. doi: 10.1016/0092-8674(83)90505-6. [DOI] [PubMed] [Google Scholar]
  16. Helmer-Citterich M., Morelli G., Macino G. Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria. EMBO J. 1983;2(8):1235–1242. doi: 10.1002/j.1460-2075.1983.tb01575.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hensgens L. A., Bonen L., de Haan M., van der Horst G., Grivell L. A. Two intron sequences in yeast mitochondrial COX1 gene: homology among URF-containing introns and strain-dependent variation in flanking exons. Cell. 1983 Feb;32(2):379–389. doi: 10.1016/0092-8674(83)90457-9. [DOI] [PubMed] [Google Scholar]
  18. Hudspeth M. E., Ainley W. M., Shumard D. S., Butow R. A., Grossman L. I. Location and structure of the var1 gene on yeast mitochondrial DNA: nucleotide sequence of the 40.0 allele. Cell. 1982 Sep;30(2):617–626. doi: 10.1016/0092-8674(82)90258-6. [DOI] [PubMed] [Google Scholar]
  19. Jacq C., Lazowska J., Slonimski P. P. Sur un nouveau mécanisme de la régulation de l'expression génétique. C R Seances Acad Sci D. 1980 Jan 14;290(2):89–92. [PubMed] [Google Scholar]
  20. Labouesse M., Slonimski P. P. Construction of novel cytochrome b genes in yeast mitochondria by subtraction or addition of introns. EMBO J. 1983;2(2):269–276. doi: 10.1002/j.1460-2075.1983.tb01416.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lazowska J., Jacq C., Slonimski P. P. Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron. Cell. 1980 Nov;22(2 Pt 2):333–348. doi: 10.1016/0092-8674(80)90344-x. [DOI] [PubMed] [Google Scholar]
  22. Leplatois P., Danchin A. Vectors for high conditional expression of cloned genes. Biochimie. 1983 Jun;65(6):317–324. doi: 10.1016/s0300-9084(83)80153-9. [DOI] [PubMed] [Google Scholar]
  23. Michel F., Jacquier A., Dujon B. Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure. Biochimie. 1982 Oct;64(10):867–881. doi: 10.1016/s0300-9084(82)80349-0. [DOI] [PubMed] [Google Scholar]
  24. Netter P., Jacq C., Carignani G., Slonimski P. P. Critical sequences within mitochondrial introns: cis-dominant mutations of the "cytochrome-b-like" intron of the oxidase gene. Cell. 1982 Apr;28(4):733–738. doi: 10.1016/0092-8674(82)90052-6. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Waring R. B., Davies R. W., Scazzocchio C., Brown T. A. Internal structure of a mitochondrial intron of Aspergillus nidulans. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6332–6336. doi: 10.1073/pnas.79.20.6332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weiss-Brummer B., Holl J., Schweyen R. J., Rödel G., Kaudewitz F. Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion. Cell. 1983 May;33(1):195–202. doi: 10.1016/0092-8674(83)90348-3. [DOI] [PubMed] [Google Scholar]
  29. Weiss-Brummer B., Rödel G., Schweyen R. J., Kaudewitz F. Expression of the split gene cob in yeast: evidence for a precursor of a "maturase" protein translated from intron 4 and preceding exons. Cell. 1982 Jun;29(2):527–536. doi: 10.1016/0092-8674(82)90169-6. [DOI] [PubMed] [Google Scholar]

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