Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Dec;15(12):6971–6978. doi: 10.1128/mcb.15.12.6971

Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein.

A S Lewin 1, J Thomas Jr 1, H K Tirupati 1
PMCID: PMC230952  PMID: 8524264

Abstract

The nuclear CBP2 gene encodes a protein essential for the splicing of a mitochondrial group I intron in Saccharomyces cerevisiae. This intron (bI5) is spliced autocatalytically in the presence of high concentrations of magnesium and monovalent salt but requires the Cbp2 protein for splicing under physiological conditions. Addition of Cbp2 during RNA synthesis permitted cotranscriptional splicing. Splicing did not occur in the transcription buffer in the absence of synthesis. The Cbp2 protein appeared to modify the folding of the intron during RNA synthesis: pause sites for RNA polymerase were altered in the presence of the protein, and some mutant transcripts that did not splice after transcription did so during transcription in the presence of Cbp2. Cotranscriptional splicing also reduced hydrolysis at the 3' splice junction. These results suggest that Cbp2 modulates the sequential folding of the ribozyme during its synthesis. In addition, splicing during transcription led to an increase in RNA synthesis with both T7 RNA polymerase and mitochondrial RNA polymerase, implying a functional coupling between transcription and splicing.

Full Text

The Full Text of this article is available as a PDF (510.4 KB).

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. Anderson J. T., Paddy M. R., Swanson M. S. PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Oct;13(10):6102–6113. doi: 10.1128/mcb.13.10.6102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banroques J., Delahodde A., Jacq C. A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing. Cell. 1986 Sep 12;46(6):837–844. doi: 10.1016/0092-8674(86)90065-6. [DOI] [PubMed] [Google Scholar]
  4. Baurén G., Wieslander L. Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription. Cell. 1994 Jan 14;76(1):183–192. doi: 10.1016/0092-8674(94)90182-1. [DOI] [PubMed] [Google Scholar]
  5. Bevilacqua P. C., Kierzek R., Johnson K. A., Turner D. H. Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods. Science. 1992 Nov 20;258(5086):1355–1358. doi: 10.1126/science.1455230. [DOI] [PubMed] [Google Scholar]
  6. Beyer A. L., Osheim Y. N. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 1988 Jun;2(6):754–765. doi: 10.1101/gad.2.6.754. [DOI] [PubMed] [Google Scholar]
  7. Buchman A. R., Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. doi: 10.1128/mcb.8.10.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bujard H., Gentz R., Lanzer M., Stueber D., Mueller M., Ibrahimi I., Haeuptle M. T., Dobberstein B. A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol. 1987;155:416–433. doi: 10.1016/0076-6879(87)55028-5. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Cech T. R. Conserved sequences and structures of group I introns: building an active site for RNA catalysis--a review. Gene. 1988 Dec 20;73(2):259–271. doi: 10.1016/0378-1119(88)90492-1. [DOI] [PubMed] [Google Scholar]
  12. Chan C. L., Landick R. Dissection of the his leader pause site by base substitution reveals a multipartite signal that includes a pause RNA hairpin. J Mol Biol. 1993 Sep 5;233(1):25–42. doi: 10.1006/jmbi.1993.1482. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Christianson T., Rabinowitz M. Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase. J Biol Chem. 1983 Nov 25;258(22):14025–14033. [PubMed] [Google Scholar]
  15. Couture S., Ellington A. D., Gerber A. S., Cherry J. M., Doudna J. A., Green R., Hanna M., Pace U., Rajagopal J., Szostak J. W. Mutational analysis of conserved nucleotides in a self-splicing group I intron. J Mol Biol. 1990 Oct 5;215(3):345–358. doi: 10.1016/s0022-2836(05)80356-0. [DOI] [PubMed] [Google Scholar]
  16. Docherty R., Rickwood D. Characterisation of RNA polymerase activity in yeast mitochondria. Eur J Biochem. 1986 Apr 1;156(1):185–188. doi: 10.1111/j.1432-1033.1986.tb09565.x. [DOI] [PubMed] [Google Scholar]
  17. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galloway Salvo J. L., Coetzee T., Belfort M. Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron. Analysis of the P6-L6a region. J Mol Biol. 1990 Feb 5;211(3):537–549. doi: 10.1016/0022-2836(90)90264-m. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Held W. A., Mizushima S., Nomura M. Reconstitution of Escherichia coli 30 S ribosomal subunits from purified molecular components. J Biol Chem. 1973 Aug 25;248(16):5720–5730. [PubMed] [Google Scholar]
  24. Herschlag D. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme. Biochemistry. 1992 Feb 11;31(5):1386–1399. doi: 10.1021/bi00120a015. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Huang M. T., Gorman C. M. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 1990 Feb 25;18(4):937–947. doi: 10.1093/nar/18.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Inoue T., Sullivan F. X., Cech T. R. New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing. J Mol Biol. 1986 May 5;189(1):143–165. doi: 10.1016/0022-2836(86)90387-6. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Jang S. H., Jaehning J. A. The yeast mitochondrial RNA polymerase specificity factor, MTF1, is similar to bacterial sigma factors. J Biol Chem. 1991 Nov 25;266(33):22671–22677. [PubMed] [Google Scholar]
  30. Jiménez-García L. F., Spector D. L. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell. 1993 Apr 9;73(1):47–59. doi: 10.1016/0092-8674(93)90159-n. [DOI] [PubMed] [Google Scholar]
  31. Kelly J. L., Lehman I. R. Yeast mitochondrial RNA polymerase. Purification and properties of the catalytic subunit. J Biol Chem. 1986 Aug 5;261(22):10340–10347. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Lazowska J., Claisse M., Gargouri A., Kotylak Z., Spyridakis A., Slonimski P. P. Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase. Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships. J Mol Biol. 1989 Jan 20;205(2):275–289. doi: 10.1016/0022-2836(89)90341-0. [DOI] [PubMed] [Google Scholar]
  35. Luehrsen K. R., Walbot V. Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol Gen Genet. 1991 Jan;225(1):81–93. doi: 10.1007/BF00282645. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. McKee E. E., Poyton R. O. Mitochondrial gene expression in saccharomyces cerevisiae. I. Optimal conditions for protein synthesis in isolated mitochondria. J Biol Chem. 1984 Jul 25;259(14):9320–9331. [PubMed] [Google Scholar]
  39. 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]
  40. Mills D. R., Dobkin C., Kramer F. R. Template-determined, variable rate of RNA chain elongation. Cell. 1978 Oct;15(2):541–550. doi: 10.1016/0092-8674(78)90022-3. [DOI] [PubMed] [Google Scholar]
  41. Mohr G., Zhang A., Gianelos J. A., Belfort M., Lambowitz A. M. The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core. Cell. 1992 May 1;69(3):483–494. doi: 10.1016/0092-8674(92)90449-m. [DOI] [PubMed] [Google Scholar]
  42. Mougey E. B., O'Reilly M., Osheim Y., Miller O. L., Jr, Beyer A., Sollner-Webb B. The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes. Genes Dev. 1993 Aug;7(8):1609–1619. doi: 10.1101/gad.7.8.1609. [DOI] [PubMed] [Google Scholar]
  43. Noller H. F., Hoffarth V., Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science. 1992 Jun 5;256(5062):1416–1419. doi: 10.1126/science.1604315. [DOI] [PubMed] [Google Scholar]
  44. Osheim Y. N., Miller O. L., Jr, Beyer A. L. RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell. 1985 Nov;43(1):143–151. doi: 10.1016/0092-8674(85)90019-4. [DOI] [PubMed] [Google Scholar]
  45. Osinga K. A., De Vries E., Van der Horst G., Tabak H. F. Processing of yeast mitochondrial messenger RNAs at a conserved dodecamer sequence. EMBO J. 1984 Apr;3(4):829–834. doi: 10.1002/j.1460-2075.1984.tb01892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Partono S., Lewin A. S. Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8192–8196. doi: 10.1073/pnas.87.21.8192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  49. Perlman P. S. Genetic analysis of RNA splicing in yeast mitochondria. Methods Enzymol. 1990;181:539–558. doi: 10.1016/0076-6879(90)81150-s. [DOI] [PubMed] [Google Scholar]
  50. Richardson J. P. Transcription termination. Crit Rev Biochem Mol Biol. 1993;28(1):1–30. doi: 10.3109/10409239309082571. [DOI] [PubMed] [Google Scholar]
  51. Ritchings B. W., Lewin A. S. Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron. Nucleic Acids Res. 1992 May 11;20(9):2349–2353. doi: 10.1093/nar/20.9.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  53. Rudolph H., Koenig-Rauseo I., Hinnen A. One-step gene replacement in yeast by cotransformation. Gene. 1985;36(1-2):87–95. doi: 10.1016/0378-1119(85)90072-1. [DOI] [PubMed] [Google Scholar]
  54. Röhl R., Nierhaus K. H. Assembly map of the large subunit (50S) of Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1982 Feb;79(3):729–733. doi: 10.1073/pnas.79.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. Ullu E., Matthews K. R., Tschudi C. Temporal order of RNA-processing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts. Mol Cell Biol. 1993 Jan;13(1):720–725. doi: 10.1128/mcb.13.1.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Walstrum S. A., Uhlenbeck O. C. The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification. Biochemistry. 1990 Nov 20;29(46):10573–10576. doi: 10.1021/bi00498a022. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. 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]
  61. Winkley C. S., Keller M. J., Jaehning J. A. A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae. J Biol Chem. 1985 Nov 15;260(26):14214–14223. [PubMed] [Google Scholar]
  62. Zarrinkar P. P., Williamson J. R. Kinetic intermediates in RNA folding. Science. 1994 Aug 12;265(5174):918–924. doi: 10.1126/science.8052848. [DOI] [PubMed] [Google Scholar]
  63. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. de Zamaroczy M., Bernardi G. Sequence organization of the mitochondrial genome of yeast--a review. Gene. 1985;37(1-3):1–17. doi: 10.1016/0378-1119(85)90252-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES