Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Jun;9(6):2765–2770. doi: 10.1128/mcb.9.6.2765

Splicing of a yeast intron containing an unusual 5' junction sequence.

M R Hodge 1, M G Cumsky 1
PMCID: PMC362354  PMID: 2548089

Abstract

The Saccharomyces cerevisiae COX5b gene contains a small intron that is unique in two respects. First, it interrupts the ATG codon that initiates translation of the COX5b product. Second, it contains a sequence at the 5' splice junction (5'-GCATGT-3') that differs from the highly conserved yeast hexanucleotide (5'-GTAPyGT-3') and from the 5'-GT found at the corresponding position in nearly all introns of eucaryotic protein-coding genes. We have analyzed both the transcripts derived from the COX5b gene and the splicing of its intron. We show here that an unspliced mRNA precursor constituted a minor fraction of the total COX5b message, even when the gene was overexpressed. We also show that both major transcripts derived from COX5b had been spliced. Our results suggest that at least in the case of COX5b, a 5'-GC can function as efficiently as the highly conserved 5'-GT in the splicing reaction.

Full text

PDF
2765

Images in this article

2768Image
on p.2768
2768Image
on p.2768
2768Image
on p.2768

Selected References

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

  1. 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]
  2. Coruzzi G., Tzagoloff A. Assembly of the mitochondrial membrane system. DNA sequence of subunit 2 of yeast cytochrome oxidase. J Biol Chem. 1979 Sep 25;254(18):9324–9330. [PubMed] [Google Scholar]
  3. Cumsky M. G., Ko C., Trueblood C. E., Poyton R. O. Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2235–2239. doi: 10.1073/pnas.82.8.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cumsky M. G., McEwen J. E., Ko C., Poyton R. O. Nuclear genes for mitochondrial proteins. Identification and isolation of a structural gene for subunit V of yeast cytochrome c oxidase. J Biol Chem. 1983 Nov 25;258(22):13418–13421. [PubMed] [Google Scholar]
  5. Cumsky M. G., Trueblood C. E., Ko C., Poyton R. O. Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V. Mol Cell Biol. 1987 Oct;7(10):3511–3519. doi: 10.1128/mcb.7.10.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dodgson J. B., Engel J. D. The nucleotide sequence of the adult chicken alpha-globin genes. J Biol Chem. 1983 Apr 10;258(7):4623–4629. [PubMed] [Google Scholar]
  7. Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
  8. Dush M. K., Sikela J. M., Khan S. A., Tischfield J. A., Stambrook P. J. Nucleotide sequence and organization of the mouse adenine phosphoribosyltransferase gene: presence of a coding region common to animal and bacterial phosphoribosyltransferases that has a variable intron/exon arrangement. Proc Natl Acad Sci U S A. 1985 May;82(9):2731–2735. doi: 10.1073/pnas.82.9.2731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Erbil C., Niessing J. The primary structure of the duck alpha D-globin gene: an unusual 5' splice junction sequence. EMBO J. 1983;2(8):1339–1343. doi: 10.1002/j.1460-2075.1983.tb01589.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fouser L. A., Friesen J. D. Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing. Cell. 1986 Apr 11;45(1):81–93. doi: 10.1016/0092-8674(86)90540-4. [DOI] [PubMed] [Google Scholar]
  11. Fox T. D. Five TGA "stop" codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6534–6538. doi: 10.1073/pnas.76.12.6534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Glaser S. M., Trueblood C. E., Dircks L. K., Poyton R. O., Cumsky M. G. Functional analysis of mitochondrial protein import in yeast. J Cell Biochem. 1988 Mar;36(3):275–287. doi: 10.1002/jcb.240360308. [DOI] [PubMed] [Google Scholar]
  13. Hodge M. R., Kim G., Singh K., Cumsky M. G. Inverse regulation of the yeast COX5 genes by oxygen and heme. Mol Cell Biol. 1989 May;9(5):1958–1964. doi: 10.1128/mcb.9.5.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. King C. R., Piatigorsky J. Alternative RNA splicing of the murine alpha A-crystallin gene: protein-coding information within an intron. Cell. 1983 Mar;32(3):707–712. doi: 10.1016/0092-8674(83)90056-9. [DOI] [PubMed] [Google Scholar]
  15. Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
  16. Levanon D., Lieman-Hurwitz J., Dafni N., Wigderson M., Sherman L., Bernstein Y., Laver-Rudich Z., Danciger E., Stein O., Groner Y. Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase. EMBO J. 1985 Jan;4(1):77–84. doi: 10.1002/j.1460-2075.1985.tb02320.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maarse A. C., Van Loon A. P., Riezman H., Gregor I., Schatz G., Grivell L. A. Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein. EMBO J. 1984 Dec 1;3(12):2831–2837. doi: 10.1002/j.1460-2075.1984.tb02216.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McEwen J. E., Cumsky M. G., Ko C., Power S. D., Poyton R. O. Mitochondrial membrane biogenesis: characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase. J Cell Biochem. 1984;24(3):229–242. doi: 10.1002/jcb.240240305. [DOI] [PubMed] [Google Scholar]
  19. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Newman A. J., Lin R. J., Cheng S. C., Abelson J. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell. 1985 Aug;42(1):335–344. doi: 10.1016/s0092-8674(85)80129-x. [DOI] [PubMed] [Google Scholar]
  21. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  22. Parker R., Guthrie C. A point mutation in the conserved hexanucleotide at a yeast 5' splice junction uncouples recognition, cleavage, and ligation. Cell. 1985 May;41(1):107–118. doi: 10.1016/0092-8674(85)90065-0. [DOI] [PubMed] [Google Scholar]
  23. Patterson T. E., Poyton R. O. COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini. J Biol Chem. 1986 Dec 25;261(36):17192–17197. [PubMed] [Google Scholar]
  24. Pikielny C. W., Rosbash M. mRNA splicing efficiency in yeast and the contribution of nonconserved sequences. Cell. 1985 May;41(1):119–126. doi: 10.1016/0092-8674(85)90066-2. [DOI] [PubMed] [Google Scholar]
  25. Rosbash M., Harris P. K., Woolford J. L., Jr, Teem J. L. The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell. 1981 Jun;24(3):679–686. doi: 10.1016/0092-8674(81)90094-5. [DOI] [PubMed] [Google Scholar]
  26. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  27. Rymond B. C., Rosbash M. Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing. Nature. 1985 Oct 24;317(6039):735–737. doi: 10.1038/317735a0. [DOI] [PubMed] [Google Scholar]
  28. Rymond B. C., Torrey D. D., Rosbash M. A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae. Genes Dev. 1987 May;1(3):238–246. doi: 10.1101/gad.1.3.238. [DOI] [PubMed] [Google Scholar]
  29. Shapiro M. B., Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987 Sep 11;15(17):7155–7174. doi: 10.1093/nar/15.17.7155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Teem J. L., Abovich N., Kaufer N. F., Schwindinger W. F., Warner J. R., Levy A., Woolford J., Leer R. J., van Raamsdonk-Duin M. M., Mager W. H. A comparison of yeast ribosomal protein gene DNA sequences. Nucleic Acids Res. 1984 Nov 26;12(22):8295–8312. doi: 10.1093/nar/12.22.8295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Trueblood C. E., Poyton R. O. Differential effectiveness of yeast cytochrome c oxidase subunit genes results from differences in expression not function. Mol Cell Biol. 1987 Oct;7(10):3520–3526. doi: 10.1128/mcb.7.10.3520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vijayraghavan U., Parker R., Tamm J., Iimura Y., Rossi J., Abelson J., Guthrie C. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 1986 Jul;5(7):1683–1695. doi: 10.1002/j.1460-2075.1986.tb04412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Warner J. R., Mitra G., Schwindinger W. F., Studeny M., Fried H. M. Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover. Mol Cell Biol. 1985 Jun;5(6):1512–1521. doi: 10.1128/mcb.5.6.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wright R. M., Dircks L. K., Poyton R. O. Characterization of COX9, the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa. Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme. J Biol Chem. 1986 Dec 25;261(36):17183–17191. [PubMed] [Google Scholar]
  37. Wright R. M., Ko C., Cumsky M. G., Poyton R. O. Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae. J Biol Chem. 1984 Dec 25;259(24):15401–15407. [PubMed] [Google Scholar]
  38. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES