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. 1990 Nov;87(21):8192–8196. doi: 10.1073/pnas.87.21.8192

Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.

S Partono 1, A S Lewin 1
PMCID: PMC54921  PMID: 2236031

Abstract

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.

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Selected References

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

  1. Barfod E. T., Cech T. R. The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing. Mol Cell Biol. 1989 Sep;9(9):3657–3666. doi: 10.1128/mcb.9.9.3657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Been M. D., Cech T. R. Sites of circularization of the Tetrahymena rRNA IVS are determined by sequence and influenced by position and secondary structure. Nucleic Acids Res. 1985 Dec 9;13(23):8389–8408. doi: 10.1093/nar/13.23.8389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burke J. M., Belfort M., Cech T. R., Davies R. W., Schweyen R. J., Shub D. A., Szostak J. W., Tabak H. F. Structural conventions for group I introns. Nucleic Acids Res. 1987 Sep 25;15(18):7217–7221. doi: 10.1093/nar/15.18.7217. [DOI] [PMC free article] [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. Burke J. M. Selection of the 3'-splice site in group I introns. FEBS Lett. 1989 Jul 3;250(2):129–133. doi: 10.1016/0014-5793(89)80704-5. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chu F. K., Maley G. F., West D. K., Belfort M., Maley F. Characterization of the intron in the phage T4 thymidylate synthase gene and evidence for its self-excision from the primary transcript. Cell. 1986 Apr 25;45(2):157–166. doi: 10.1016/0092-8674(86)90379-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Doudna J. A., Cormack B. P., Szostak J. W. RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7402–7406. doi: 10.1073/pnas.86.19.7402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Hall D. H., Povinelli C. M., Ehrenman K., Pedersen-Lane J., Chu F., Belfort M. Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis. Cell. 1987 Jan 16;48(1):63–71. doi: 10.1016/0092-8674(87)90356-4. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Michel F., Dujon B. Conservation of RNA secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members. EMBO J. 1983;2(1):33–38. doi: 10.1002/j.1460-2075.1983.tb01376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Michel F., Hanna M., Green R., Bartel D. P., Szostak J. W. The guanosine binding site of the Tetrahymena ribozyme. Nature. 1989 Nov 23;342(6248):391–395. doi: 10.1038/342391a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Price J. V., Cech T. R. Determinants of the 3' splice site for self-splicing of the Tetrahymena pre-rRNA. Genes Dev. 1988 Nov;2(11):1439–1447. doi: 10.1101/gad.2.11.1439. [DOI] [PubMed] [Google Scholar]
  23. Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suh E. R., Waring R. B. Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron. Mol Cell Biol. 1990 Jun;10(6):2960–2965. doi: 10.1128/mcb.10.6.2960. [DOI] [PMC free article] [PubMed] [Google Scholar]

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