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. 1989 Apr;9(4):1526–1535. doi: 10.1128/mcb.9.4.1526

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

K B Gatermann 1, A Hoffmann 1, G H Rosenberg 1, N F Käufer 1
PMCID: PMC362569  PMID: 2725514

Abstract

Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product. This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene. Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly. A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site. No functional product is produced from this transcript. An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product. Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns. We discuss the possibility that S. pombe introns represent a simple type of eucaryotic 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. Aebi M., Hornig H., Weissmann C. 5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU. Cell. 1987 Jul 17;50(2):237–246. doi: 10.1016/0092-8674(87)90219-4. [DOI] [PubMed] [Google Scholar]
  2. Bach M. L. Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe. Curr Genet. 1987;12(7):527–534. doi: 10.1007/BF00419562. [DOI] [PubMed] [Google Scholar]
  3. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  4. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  5. Chabot B., Steitz J. A. Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes. Mol Cell Biol. 1987 Jan;7(1):281–293. doi: 10.1128/mcb.7.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  7. Couto J. R., Tamm J., Parker R., Guthrie C. A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation. Genes Dev. 1987 Jul;1(5):445–455. doi: 10.1101/gad.1.5.445. [DOI] [PubMed] [Google Scholar]
  8. Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
  9. Duchêne M., Löw A., Schweizer A., Domdey H. Molecular consequences of truncations of the first exon for in vitro splicing of yeast actin pre-mRNA. Nucleic Acids Res. 1988 Aug 11;16(15):7233–7239. doi: 10.1093/nar/16.15.7233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fink G. R. Pseudogenes in yeast? Cell. 1987 Apr 10;49(1):5–6. doi: 10.1016/0092-8674(87)90746-x. [DOI] [PubMed] [Google Scholar]
  11. Fouser L. A., Friesen J. D. Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing. Cell. 1986 Apr 11;45(1):81–93. doi: 10.1016/0092-8674(86)90540-4. [DOI] [PubMed] [Google Scholar]
  12. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  13. Gaterman K. B., Rosenberg G. H., Käufer N. F. Double-stranded sequencing, using mini-prep plasmids, in eleven hours. Biotechniques. 1988 Nov-Dec;6(10):951–952. [PubMed] [Google Scholar]
  14. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  15. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Holm C., Meeks-Wagner D. W., Fangman W. L., Botstein D. A rapid, efficient method for isolating DNA from yeast. Gene. 1986;42(2):169–173. doi: 10.1016/0378-1119(86)90293-3. [DOI] [PubMed] [Google Scholar]
  18. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klinz F. J., Gallwitz D. Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1985 Jun 11;13(11):3791–3804. doi: 10.1093/nar/13.11.3791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Konarska M. M., Sharp P. A. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986 Sep 12;46(6):845–855. doi: 10.1016/0092-8674(86)90066-8. [DOI] [PubMed] [Google Scholar]
  21. Kretzner L., Rymond B. C., Rosbash M. S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA. Cell. 1987 Aug 14;50(4):593–602. doi: 10.1016/0092-8674(87)90032-8. [DOI] [PubMed] [Google Scholar]
  22. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Käufer N. F., Fried H. M., Schwindinger W. F., Jasin M., Warner J. R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 1983 May 25;11(10):3123–3135. doi: 10.1093/nar/11.10.3123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Käufer N. F., Simanis V., Nurse P. Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence. Nature. 1985 Nov 7;318(6041):78–80. doi: 10.1038/318078a0. [DOI] [PubMed] [Google Scholar]
  25. Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
  26. Lossky M., Anderson G. J., Jackson S. P., Beggs J. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions. Cell. 1987 Dec 24;51(6):1019–1026. doi: 10.1016/0092-8674(87)90588-5. [DOI] [PubMed] [Google Scholar]
  27. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  28. Mertins P., Gallwitz D. Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element. EMBO J. 1987 Jun;6(6):1757–1763. doi: 10.1002/j.1460-2075.1987.tb02428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  31. Newman A. J., Lin R. J., Cheng S. C., Abelson J. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell. 1985 Aug;42(1):335–344. doi: 10.1016/s0092-8674(85)80129-x. [DOI] [PubMed] [Google Scholar]
  32. Nischt R., Thüroff E., Küfer N. F. Molecular cloning of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe. Curr Genet. 1986;10(5):365–370. doi: 10.1007/BF00418408. [DOI] [PubMed] [Google Scholar]
  33. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  34. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  35. Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
  36. Rao R. N., Rogers S. G. Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene. 1979 Sep;7(1):79–82. doi: 10.1016/0378-1119(79)90044-1. [DOI] [PubMed] [Google Scholar]
  37. Rautmann G., Matthes H. W., Gait M. J., Breathnach R. Synthetic donor and acceptor splice sites function in an RNA polymerase B (II) transcription unit. EMBO J. 1984 Sep;3(9):2021–2028. doi: 10.1002/j.1460-2075.1984.tb02085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reed R., Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. doi: 10.1016/0092-8674(86)90343-0. [DOI] [PubMed] [Google Scholar]
  39. Rodriguez J. R., Pikielny C. W., Rosbash M. In vivo characterization of yeast mRNA processing intermediates. Cell. 1984 Dec;39(3 Pt 2):603–610. doi: 10.1016/0092-8674(84)90467-7. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  44. Swida U., Thüroff E., Steinert E., Käufer N. F. A non-conserved sequence in the 5'region of the CYH2 intron from Saccharomyces cerevisiae controls splicing efficiency of the pre-mRNA. Yeast. 1988 Sep;4(3):209–217. doi: 10.1002/yea.320040306. [DOI] [PubMed] [Google Scholar]
  45. Takeda T., Yamamoto M. Analysis and in vivo disruption of the gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3580–3584. doi: 10.1073/pnas.84.11.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Uemura T., Morino K., Uzawa S., Shiozaki K., Yanagida M. Cloning and sequencing of Schizosaccharomyces pombe DNA topoisomerase I gene, and effect of gene disruption. Nucleic Acids Res. 1987 Dec 10;15(23):9727–9739. doi: 10.1093/nar/15.23.9727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vijayraghavan U., Parker R., Tamm J., Iimura Y., Rossi J., Abelson J., Guthrie C. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 1986 Jul;5(7):1683–1695. doi: 10.1002/j.1460-2075.1986.tb04412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wright A., Maundrell K., Heyer W. D., Beach D., Nurse P. Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Plasmid. 1986 Mar;15(2):156–158. doi: 10.1016/0147-619x(86)90051-x. [DOI] [PubMed] [Google Scholar]
  49. Zeitlin S., Efstratiadis A. In vivo splicing products of the rabbit beta-globin pre-mRNA. Cell. 1984 Dec;39(3 Pt 2):589–602. doi: 10.1016/0092-8674(84)90466-5. [DOI] [PubMed] [Google Scholar]
  50. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]

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