Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Nov 15;25(22):4658–4665. doi: 10.1093/nar/25.22.4658

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

C M Romfo 1, J A Wise 1
PMCID: PMC147086  PMID: 9358179

Abstract

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.

Full Text

The Full Text of this article is available as a PDF (387.6 KB).

Selected References

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

  1. Abovich N., Liao X. C., Rosbash M. The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition. Genes Dev. 1994 Apr 1;8(7):843–854. doi: 10.1101/gad.8.7.843. [DOI] [PubMed] [Google Scholar]
  2. Aebi M., Hornig H., Padgett R. A., Reiser J., Weissmann C. Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA. Cell. 1986 Nov 21;47(4):555–565. doi: 10.1016/0092-8674(86)90620-3. [DOI] [PubMed] [Google Scholar]
  3. Alvarez C. J., Romfo C. M., Vanhoy R. W., Porter G. L., Wise J. A. Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo. RNA. 1996 May;2(5):404–418. [PMC free article] [PubMed] [Google Scholar]
  4. Blumenthal T., Thomas J. Cis and trans mRNA splicing in C. elegans. Trends Genet. 1988 Nov;4(11):305–308. doi: 10.1016/0168-9525(88)90107-2. [DOI] [PubMed] [Google Scholar]
  5. Brennwald P., Liao X., Holm K., Porter G., Wise J. A. Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle. Mol Cell Biol. 1988 Apr;8(4):1580–1590. doi: 10.1128/mcb.8.4.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cellini A., Felder E., Rossi J. J. Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3' splice site. EMBO J. 1986 May;5(5):1023–1030. doi: 10.1002/j.1460-2075.1986.tb04317.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Champion-Arnaud P., Gozani O., Palandjian L., Reed R. Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations. Mol Cell Biol. 1995 Oct;15(10):5750–5756. doi: 10.1128/mcb.15.10.5750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chiara M. D., Gozani O., Bennett M., Champion-Arnaud P., Palandjian L., Reed R. Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly. Mol Cell Biol. 1996 Jul;16(7):3317–3326. doi: 10.1128/mcb.16.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coolidge C. J., Seely R. J., Patton J. G. Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nucleic Acids Res. 1997 Feb 15;25(4):888–896. doi: 10.1093/nar/25.4.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Csank C., Taylor F. M., Martindale D. W. Nuclear pre-mRNA introns: analysis and comparison of intron sequences from Tetrahymena thermophila and other eukaryotes. Nucleic Acids Res. 1990 Sep 11;18(17):5133–5141. doi: 10.1093/nar/18.17.5133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Fields C. Information content of Caenorhabditis elegans splice site sequences varies with intron length. Nucleic Acids Res. 1990 Mar 25;18(6):1509–1512. doi: 10.1093/nar/18.6.1509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  14. Hindley J., Phear G. A. Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene. 1984 Nov;31(1-3):129–134. doi: 10.1016/0378-1119(84)90203-8. [DOI] [PubMed] [Google Scholar]
  15. Hiraoka Y., Toda T., Yanagida M. The NDA3 gene of fission yeast encodes beta-tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell. 1984 Dec;39(2 Pt 1):349–358. doi: 10.1016/0092-8674(84)90013-8. [DOI] [PubMed] [Google Scholar]
  16. Kennedy C. F., Berget S. M. Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron. Mol Cell Biol. 1997 May;17(5):2774–2780. doi: 10.1128/mcb.17.5.2774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990 Jul 5;265(19):10857–10864. [PubMed] [Google Scholar]
  18. Mount S. M., Burks C., Hertz G., Stormo G. D., White O., Fields C. Splicing signals in Drosophila: intron size, information content, and consensus sequences. Nucleic Acids Res. 1992 Aug 25;20(16):4255–4262. doi: 10.1093/nar/20.16.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nelson K. K., Green M. R. Mammalian U2 snRNP has a sequence-specific RNA-binding activity. Genes Dev. 1989 Oct;3(10):1562–1571. doi: 10.1101/gad.3.10.1562. [DOI] [PubMed] [Google Scholar]
  20. Patterson B., Guthrie C. A U-rich tract enhances usage of an alternative 3' splice site in yeast. Cell. 1991 Jan 11;64(1):181–187. doi: 10.1016/0092-8674(91)90219-o. [DOI] [PubMed] [Google Scholar]
  21. Porter G., Brennwald P., Wise J. A. U1 small nuclear RNA from Schizosaccharomyces pombe has unique and conserved features and is encoded by an essential single-copy gene. Mol Cell Biol. 1990 Jun;10(6):2874–2881. doi: 10.1128/mcb.10.6.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Potashkin J., Naik K., Wentz-Hunter K. U2AF homolog required for splicing in vivo. Science. 1993 Oct 22;262(5133):573–575. doi: 10.1126/science.8211184. [DOI] [PubMed] [Google Scholar]
  23. Prabhala G., Rosenberg G. H., Käufer N. F. Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe. Yeast. 1992 Mar;8(3):171–182. doi: 10.1002/yea.320080303. [DOI] [PubMed] [Google Scholar]
  24. Reed R., Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. doi: 10.1016/0092-8674(85)90064-9. [DOI] [PubMed] [Google Scholar]
  25. Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
  26. Reich C. I., VanHoy R. W., Porter G. L., Wise J. A. Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast. Cell. 1992 Jun 26;69(7):1159–1169. doi: 10.1016/0092-8674(92)90637-r. [DOI] [PubMed] [Google Scholar]
  27. Roscigno R. F., Weiner M., Garcia-Blanco M. A. A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing. J Biol Chem. 1993 May 25;268(15):11222–11229. [PubMed] [Google Scholar]
  28. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  29. Ruskin B., Green M. R. Role of the 3' splice site consensus sequence in mammalian pre-mRNA splicing. Nature. 1985 Oct 24;317(6039):732–734. doi: 10.1038/317732a0. [DOI] [PubMed] [Google Scholar]
  30. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  31. Russell P., Nurse P. Schizosaccharomyces pombe and Saccharomyces cerevisiae: a look at yeasts divided. Cell. 1986 Jun 20;45(6):781–782. doi: 10.1016/0092-8674(86)90550-7. [DOI] [PubMed] [Google Scholar]
  32. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  33. Séraphin B., Kandels-Lewis S. 3' splice site recognition in S. cerevisiae does not require base pairing with U1 snRNA. Cell. 1993 May 21;73(4):803–812. doi: 10.1016/0092-8674(93)90258-r. [DOI] [PubMed] [Google Scholar]
  34. Umen J. G., Guthrie C. A novel role for a U5 snRNP protein in 3' splice site selection. Genes Dev. 1995 Apr 1;9(7):855–868. doi: 10.1101/gad.9.7.855. [DOI] [PubMed] [Google Scholar]
  35. Wentz-Hunter K., Potashkin J. The small subunit of the splicing factor U2AF is conserved in fission yeast. Nucleic Acids Res. 1996 May 15;24(10):1849–1854. doi: 10.1093/nar/24.10.1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wise J. A. Preparation and analysis of low molecular weight RNAs and small ribonucleoproteins. Methods Enzymol. 1991;194:405–415. doi: 10.1016/0076-6879(91)94031-7. [DOI] [PubMed] [Google Scholar]
  37. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]
  38. Zhang H., Blumenthal T. Functional analysis of an intron 3' splice site in Caenorhabditis elegans. RNA. 1996 Apr;2(4):380–388. [PMC free article] [PubMed] [Google Scholar]
  39. Zhang M. Q., Marr T. G. Fission yeast gene structure and recognition. Nucleic Acids Res. 1994 May 11;22(9):1750–1759. doi: 10.1093/nar/22.9.1750. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES