Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Dec;9(12):5424–5433. doi: 10.1128/mcb.9.12.5424

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

A Gampel 1, M Nishikimi 1, A Tzagoloff 1
PMCID: PMC363710  PMID: 2685564

Abstract

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.

Full text

PDF
5424

Images in this article

5426Image
on p.5426
5427Image
on p.5427
5428Image
on p.5428
5428Image
on p.5428
5428Image
on p.5428
5429Image
on p.5429
5429Image
on p.5429
5430Image
on p.5430
5431Image
on p.5431

Selected References

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

  1. Akins R. A., Lambowitz A. M. A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof. Cell. 1987 Jul 31;50(3):331–345. doi: 10.1016/0092-8674(87)90488-0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Been M. D., Cech T. R. One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity. Cell. 1986 Oct 24;47(2):207–216. doi: 10.1016/0092-8674(86)90443-5. [DOI] [PubMed] [Google Scholar]
  4. Burke J. M., Irvine K. D., Kaneko K. J., Kerker B. J., Oettgen A. B., Tierney W. M., Williamson C. L., Zaug A. J., Cech T. R. Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor. Cell. 1986 Apr 25;45(2):167–176. doi: 10.1016/0092-8674(86)90380-6. [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. 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]
  7. Cech T. R. The chemistry of self-splicing RNA and RNA enzymes. Science. 1987 Jun 19;236(4808):1532–1539. doi: 10.1126/science.2438771. [DOI] [PubMed] [Google Scholar]
  8. Cech T. R., Zaug A. J., Grabowski P. J. In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell. 1981 Dec;27(3 Pt 2):487–496. doi: 10.1016/0092-8674(81)90390-1. [DOI] [PubMed] [Google Scholar]
  9. Chu F. K., Maley G. F., Belfort M., Maley F. In vitro expression of the intron-containing gene for T4 phage thymidylate synthase. J Biol Chem. 1985 Sep 5;260(19):10680–10688. [PubMed] [Google Scholar]
  10. 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]
  11. Corkey B. E., Duszynski J., Rich T. L., Matschinsky B., Williamson J. R. Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria. J Biol Chem. 1986 Feb 25;261(6):2567–2574. [PubMed] [Google Scholar]
  12. 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]
  13. Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
  14. 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]
  15. Garriga G., Lambowitz A. M., Inoue T., Cech T. R. Mechanism of recognition of the 5' splice site in self-splicing group I introns. Nature. 1986 Jul 3;322(6074):86–89. doi: 10.1038/322086a0. [DOI] [PubMed] [Google Scholar]
  16. Garriga G., Lambowitz A. M. Protein-dependent splicing of a group I intron in ribonucleoprotein particles and soluble fractions. Cell. 1986 Aug 29;46(5):669–680. doi: 10.1016/0092-8674(86)90342-9. [DOI] [PubMed] [Google Scholar]
  17. Garriga G., Lambowitz A. M. RNA splicing in neurospora mitochondria: self-splicing of a mitochondrial intron in vitro. Cell. 1984 Dec;39(3 Pt 2):631–641. doi: 10.1016/0092-8674(84)90470-7. [DOI] [PubMed] [Google Scholar]
  18. Grabowski P. J., Zaug A. J., Cech T. R. The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena. Cell. 1981 Feb;23(2):467–476. doi: 10.1016/0092-8674(81)90142-2. [DOI] [PubMed] [Google Scholar]
  19. Guerrier-Takada C., Altman S. Structure in solution of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli. Biochemistry. 1984 Dec 18;23(26):6327–6334. doi: 10.1021/bi00321a006. [DOI] [PubMed] [Google Scholar]
  20. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983 Dec;35(3 Pt 2):849–857. doi: 10.1016/0092-8674(83)90117-4. [DOI] [PubMed] [Google Scholar]
  21. Guerrier-Takada C., Haydock K., Allen L., Altman S. Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli. Biochemistry. 1986 Apr 8;25(7):1509–1515. doi: 10.1021/bi00355a006. [DOI] [PubMed] [Google Scholar]
  22. Herbert C. J., Labouesse M., Dujardin G., Slonimski P. P. The NAM2 proteins from S. cerevisiae and S. douglasii are mitochondrial leucyl-tRNA synthetases, and are involved in mRNA splicing. EMBO J. 1988 Feb;7(2):473–483. doi: 10.1002/j.1460-2075.1988.tb02835.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Inoue T., Cech T. R. Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase. Proc Natl Acad Sci U S A. 1985 Feb;82(3):648–652. doi: 10.1073/pnas.82.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Inoue T., Sullivan F. X., Cech T. R. Intermolecular exon ligation of the rRNA precursor of Tetrahymena: oligonucleotides can function as 5' exons. Cell. 1985 Dec;43(2 Pt 1):431–437. doi: 10.1016/0092-8674(85)90173-4. [DOI] [PubMed] [Google Scholar]
  26. Kreike J., Schulze M., Pillar T., Körte A., Rödel G. Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae. Curr Genet. 1986;11(3):185–191. doi: 10.1007/BF00420605. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  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. Robertson H. D., Altman S., Smith J. D. Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid presursor. J Biol Chem. 1972 Aug 25;247(16):5243–5251. [PubMed] [Google Scholar]
  35. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Williamson C. L., Tierney W. M., Kerker B. J., Burke J. M. Site-directed mutagenesis of core sequence elements 9R', 9L, 9R, and 2 in self-splicing Tetrahymena pre-rRNA. J Biol Chem. 1987 Oct 25;262(30):14672–14682. [PubMed] [Google Scholar]
  38. 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 Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES