Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Apr 15;25(8):1597–1604. doi: 10.1093/nar/25.8.1597

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

L C Shaw 1, A S Lewin 1
PMCID: PMC146636  PMID: 9092668

Abstract

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.

Full Text

The Full Text of this article is available as a PDF (206.2 KB).

Selected References

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

  1. Alexander N. J., Periman P. S., Hanson D. K., Mahler H. R. Mosaic organization of a mitochondrial gene: evidence from double mutants in the cytochrome b region of Saccharomyces cerevisiae. Cell. 1980 May;20(1):199–206. doi: 10.1016/0092-8674(80)90247-0. [DOI] [PubMed] [Google Scholar]
  2. Becker M. M., Wang Z. Origin of ultraviolet damage in DNA. J Mol Biol. 1989 Dec 5;210(3):429–438. doi: 10.1016/0022-2836(89)90120-4. [DOI] [PubMed] [Google Scholar]
  3. Been M. D., Cech T. R. RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme. Science. 1988 Mar 18;239(4846):1412–1416. doi: 10.1126/science.2450400. [DOI] [PubMed] [Google Scholar]
  4. Bertrand H., Bridge P., Collins R. A., Garriga G., Lambowitz A. M. RNA splicing in Neurospora mitochondria. Characterization of new nuclear mutants with defects in splicing the mitochondrial large rRNA. Cell. 1982 Jun;29(2):517–526. doi: 10.1016/0092-8674(82)90168-4. [DOI] [PubMed] [Google Scholar]
  5. Burke J. M. Molecular genetics of group I introns: RNA structures and protein factors required for splicing--a review. Gene. 1988 Dec 20;73(2):273–294. doi: 10.1016/0378-1119(88)90493-3. [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., Bass B. L. Biological catalysis by RNA. Annu Rev Biochem. 1986;55:599–629. doi: 10.1146/annurev.bi.55.070186.003123. [DOI] [PubMed] [Google Scholar]
  8. Cech T. R., Damberger S. H., Gutell R. R. Representation of the secondary and tertiary structure of group I introns. Nat Struct Biol. 1994 May;1(5):273–280. doi: 10.1038/nsb0594-273. [DOI] [PubMed] [Google Scholar]
  9. Cherniack A. D., Garriga G., Kittle J. D., Jr, Akins R. A., Lambowitz A. M. Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases. Cell. 1990 Aug 24;62(4):745–755. doi: 10.1016/0092-8674(90)90119-y. [DOI] [PubMed] [Google Scholar]
  10. Coetzee T., Herschlag D., Belfort M. Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones. Genes Dev. 1994 Jul 1;8(13):1575–1588. doi: 10.1101/gad.8.13.1575. [DOI] [PubMed] [Google Scholar]
  11. Collins R. A., Lambowitz A. M. RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1. J Mol Biol. 1985 Aug 5;184(3):413–428. doi: 10.1016/0022-2836(85)90291-8. [DOI] [PubMed] [Google Scholar]
  12. Conde J., Fink G. R. A mutant of Saccharomyces cerevisiae defective for nuclear fusion. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3651–3655. doi: 10.1073/pnas.73.10.3651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doetsch P. W., Zasatawny T. H., Martin A. M., Dizdaroglu M. Monomeric base damage products from adenine, guanine, and thymine induced by exposure of DNA to ultraviolet radiation. Biochemistry. 1995 Jan 24;34(3):737–742. doi: 10.1021/bi00003a005. [DOI] [PubMed] [Google Scholar]
  14. Dujon B., Bolotin-Fukuhara M., Coen D., Deutsch J., Netter P., Slonimski P. P., Weill L. Mitochondrial genetics. XI. Mutations at the mitochondrial locus omega affecting the recombination of mitochondrial genes in Saccharomyces cerevisiae. Mol Gen Genet. 1976 Jan 16;143(2):131–165. doi: 10.1007/BF00266918. [DOI] [PubMed] [Google Scholar]
  15. Gampel A., Cech T. R. Binding of the CBP2 protein to a yeast mitochondrial group I intron requires the catalytic core of the RNA. Genes Dev. 1991 Oct;5(10):1870–1880. doi: 10.1101/gad.5.10.1870. [DOI] [PubMed] [Google Scholar]
  16. Gampel A., Nishikimi M., Tzagoloff A. CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron. Mol Cell Biol. 1989 Dec;9(12):5424–5433. doi: 10.1128/mcb.9.12.5424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gampel A., Tzagoloff A. In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA. Mol Cell Biol. 1987 Jul;7(7):2545–2551. doi: 10.1128/mcb.7.7.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hill J., McGraw P., Tzagoloff A. A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene. J Biol Chem. 1985 Mar 25;260(6):3235–3238. [PubMed] [Google Scholar]
  20. Illangasekare M., Sanchez G., Nickles T., Yarus M. Aminoacyl-RNA synthesis catalyzed by an RNA. Science. 1995 Feb 3;267(5198):643–647. doi: 10.1126/science.7530860. [DOI] [PubMed] [Google Scholar]
  21. Jacquier A., Dujon B. An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell. 1985 Jun;41(2):383–394. doi: 10.1016/s0092-8674(85)80011-8. [DOI] [PubMed] [Google Scholar]
  22. Jaeger L., Westhof E., Michel F. Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA. J Mol Biol. 1991 Oct 20;221(4):1153–1164. doi: 10.1016/0022-2836(91)90925-v. [DOI] [PubMed] [Google Scholar]
  23. Labouesse M., Herbert C. J., Dujardin G., Slonimski P. P. Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene. EMBO J. 1987 Mar;6(3):713–721. doi: 10.1002/j.1460-2075.1987.tb04812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lamb M. R., Anziano P. Q., Glaus K. R., Hanson D. K., Klapper H. J., Perlman P. S., Mahler H. R. Functional domains in introns. RNA processing intermediates in cis- and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b. J Biol Chem. 1983 Feb 10;258(3):1991–1999. [PubMed] [Google Scholar]
  25. Lambowitz A. M., Perlman P. S. Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing. Trends Biochem Sci. 1990 Nov;15(11):440–444. doi: 10.1016/0968-0004(90)90283-h. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lewin A. S., Thomas J., Jr, Tirupati H. K. Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein. Mol Cell Biol. 1995 Dec;15(12):6971–6978. doi: 10.1128/mcb.15.12.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li G. Y., Tian G. L., Slonimski P. P., Herbert C. J. The CBP2 gene from Saccharomyces douglasii is a functional homologue of the Saccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome. Mol Gen Genet. 1996 Feb 25;250(3):316–322. doi: 10.1007/BF02174389. [DOI] [PubMed] [Google Scholar]
  29. Macreadie I. G., Scott R. M., Zinn A. R., Butow R. A. Transposition of an intron in yeast mitochondria requires a protein encoded by that intron. Cell. 1985 Jun;41(2):395–402. doi: 10.1016/s0092-8674(85)80012-x. [DOI] [PubMed] [Google Scholar]
  30. Majumder A. L., Akins R. A., Wilkinson J. G., Kelley R. L., Snook A. J., Lambowitz A. M. Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity. Mol Cell Biol. 1989 May;9(5):2089–2104. doi: 10.1128/mcb.9.5.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McGraw P., Tzagoloff A. Assembly of the mitochondrial membrane system. Characterization of a yeast nuclear gene involved in the processing of the cytochrome b pre-mRNA. J Biol Chem. 1983 Aug 10;258(15):9459–9468. [PubMed] [Google Scholar]
  32. Michel F., Westhof E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol. 1990 Dec 5;216(3):585–610. doi: 10.1016/0022-2836(90)90386-Z. [DOI] [PubMed] [Google Scholar]
  33. Partono S., Lewin A. S. Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria. Mol Cell Biol. 1988 Jun;8(6):2562–2571. doi: 10.1128/mcb.8.6.2562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Partono S., Lewin A. S. The rate and specificity of a group I ribozyme are inversely affected by choice of monovalent salt. Nucleic Acids Res. 1991 Feb 11;19(3):605–609. doi: 10.1093/nar/19.3.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Patel P. H., Preston B. D. Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):549–553. doi: 10.1073/pnas.91.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peliska J. A., Benkovic S. J. Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Science. 1992 Nov 13;258(5085):1112–1118. doi: 10.1126/science.1279806. [DOI] [PubMed] [Google Scholar]
  37. Piccirilli J. A., McConnell T. S., Zaug A. J., Noller H. F., Cech T. R. Aminoacyl esterase activity of the Tetrahymena ribozyme. Science. 1992 Jun 5;256(5062):1420–1424. doi: 10.1126/science.1604316. [DOI] [PubMed] [Google Scholar]
  38. Schmelzer C., Haid A., Grosch G., Schweyen R. J., Kaudewitz F. Pathways of transcript splicing in yeast mitochondria. Mutations in intervening sequences of the split gene COB reveal a requirement for intervening sequence-encoded products. J Biol Chem. 1981 Jul 25;256(14):7610–7619. [PubMed] [Google Scholar]
  39. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shaw L. C., Lewin A. S. Protein-induced folding of a group I intron in cytochrome b pre-mRNA. J Biol Chem. 1995 Sep 15;270(37):21552–21562. doi: 10.1074/jbc.270.37.21552. [DOI] [PubMed] [Google Scholar]
  41. Shaw L. C., Thomas J., Jr, Lewin A. S. The Cbp2 protein suppresses splice site mutations in a group I intron. Nucleic Acids Res. 1996 Sep 1;24(17):3415–3423. doi: 10.1093/nar/24.17.3415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  43. Séraphin B., Boulet A., Simon M., Faye G. Construction of a yeast strain devoid of mitochondrial introns and its use to screen nuclear genes involved in mitochondrial splicing. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6810–6814. doi: 10.1073/pnas.84.19.6810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Séraphin B., Simon M., Faye G. MSS18, a yeast nuclear gene involved in the splicing of intron aI5 beta of the mitochondrial cox1 transcript. EMBO J. 1988 May;7(5):1455–1464. doi: 10.1002/j.1460-2075.1988.tb02963.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weeks K. M., Cech T. R. Assembly of a ribonucleoprotein catalyst by tertiary structure capture. Science. 1996 Jan 19;271(5247):345–348. doi: 10.1126/science.271.5247.345. [DOI] [PubMed] [Google Scholar]
  46. Weeks K. M., Cech T. R. Efficient protein-facilitated splicing of the yeast mitochondrial bI5 intron. Biochemistry. 1995 Jun 13;34(23):7728–7738. doi: 10.1021/bi00023a020. [DOI] [PubMed] [Google Scholar]
  47. Weeks K. M., Cech T. R. Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain. Cell. 1995 Jul 28;82(2):221–230. doi: 10.1016/0092-8674(95)90309-7. [DOI] [PubMed] [Google Scholar]
  48. Zaug A. J., Been M. D., Cech T. R. The Tetrahymena ribozyme acts like an RNA restriction endonuclease. Nature. 1986 Dec 4;324(6096):429–433. doi: 10.1038/324429a0. [DOI] [PubMed] [Google Scholar]
  49. van der Horst G., Tabak H. F. Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors. Cell. 1985 Apr;40(4):759–766. doi: 10.1016/0092-8674(85)90335-6. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES