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. 1997 Mar 1;25(5):1028–1035. doi: 10.1093/nar/25.5.1028

Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue.

T Gross 1, M Lützelberger 1, H Weigmann 1, A Klingenhoff 1, S Shenoy 1, N F Käufer 1
PMCID: PMC146536  PMID: 9102632

Abstract

The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain.

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Selected References

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  1. Alahari S. K., Schmidt H., Käufer N. F. The fission yeast prp4+ gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth. Nucleic Acids Res. 1993 Aug 25;21(17):4079–4083. doi: 10.1093/nar/21.17.4079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amrein H., Hedley M. L., Maniatis T. The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2. Cell. 1994 Feb 25;76(4):735–746. doi: 10.1016/0092-8674(94)90512-6. [DOI] [PubMed] [Google Scholar]
  3. Ben-David Y., Letwin K., Tannock L., Bernstein A., Pawson T. A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators. EMBO J. 1991 Feb;10(2):317–325. doi: 10.1002/j.1460-2075.1991.tb07952.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bouvier D., Baldacci G. The N-terminus of fission yeast DNA polymerase alpha contains a basic pentapeptide that acts in vivo as a nuclear localization signal. Mol Biol Cell. 1995 Dec;6(12):1697–1705. doi: 10.1091/mbc.6.12.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cardinali B., Cohen P. T., Lamond A. I. Protein phosphatase 1 can modulate alternative 5' splice site selection in a HeLa splicing extract. FEBS Lett. 1994 Oct 3;352(3):276–280. doi: 10.1016/0014-5793(94)00973-2. [DOI] [PubMed] [Google Scholar]
  7. Colwill K., Pawson T., Andrews B., Prasad J., Manley J. L., Bell J. C., Duncan P. I. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 1996 Jan 15;15(2):265–275. [PMC free article] [PubMed] [Google Scholar]
  8. Cáceres J. F., Krainer A. R. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 1993 Dec;12(12):4715–4726. doi: 10.1002/j.1460-2075.1993.tb06160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  10. Fu X. D. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. doi: 10.1038/365082a0. [DOI] [PubMed] [Google Scholar]
  11. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  12. Gatermann K. B., Hoffmann A., Rosenberg G. H., Käufer N. F. Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe. Mol Cell Biol. 1989 Apr;9(4):1526–1535. doi: 10.1128/mcb.9.4.1526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gui J. F., Lane W. S., Fu X. D. A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature. 1994 Jun 23;369(6482):678–682. doi: 10.1038/369678a0. [DOI] [PubMed] [Google Scholar]
  14. Hanks S. K., Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576–596. [PubMed] [Google Scholar]
  15. Horowitz D. S., Krainer A. R. Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet. 1994 Mar;10(3):100–106. doi: 10.1016/0168-9525(94)90233-x. [DOI] [PubMed] [Google Scholar]
  16. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  17. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  18. Krämer A., Legrain P., Mulhauser F., Gröning K., Brosi R., Bilbe G. Splicing factor SF3a60 is the mammalian homologue of PRP9 of S.cerevisiae: the conserved zinc finger-like motif is functionally exchangeable in vivo. Nucleic Acids Res. 1994 Dec 11;22(24):5223–5228. doi: 10.1093/nar/22.24.5223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  21. Lamm G. M., Lamond A. I. Non-snRNP protein splicing factors. Biochim Biophys Acta. 1993 Jun 25;1173(3):247–265. doi: 10.1016/0167-4781(93)90122-t. [DOI] [PubMed] [Google Scholar]
  22. Lundgren K., Allan S., Urushiyama S., Tani T., Ohshima Y., Frendewey D., Beach D. A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase. Mol Biol Cell. 1996 Jul;7(7):1083–1094. doi: 10.1091/mbc.7.7.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Manley J. L., Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. doi: 10.1101/gad.10.13.1569. [DOI] [PubMed] [Google Scholar]
  24. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  25. Mermoud J. E., Cohen P. T., Lamond A. I. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J. 1994 Dec 1;13(23):5679–5688. doi: 10.1002/j.1460-2075.1994.tb06906.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mermoud J. E., Cohen P., Lamond A. I. Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res. 1992 Oct 25;20(20):5263–5269. doi: 10.1093/nar/20.20.5263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  28. Morgan D. O., De Bondt H. L. Protein kinase regulation: insights from crystal structure analysis. Curr Opin Cell Biol. 1994 Apr;6(2):239–246. doi: 10.1016/0955-0674(94)90142-2. [DOI] [PubMed] [Google Scholar]
  29. Newman A. Small nuclear RNAs and pre-mRNA splicing. Curr Opin Cell Biol. 1994 Jun;6(3):360–367. doi: 10.1016/0955-0674(94)90027-2. [DOI] [PubMed] [Google Scholar]
  30. Reed R. Initial splice-site recognition and pairing during pre-mRNA splicing. Curr Opin Genet Dev. 1996 Apr;6(2):215–220. doi: 10.1016/s0959-437x(96)80053-0. [DOI] [PubMed] [Google Scholar]
  31. Rosenberg G. H., Alahari S. K., Käufer N. F. prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns. Mol Gen Genet. 1991 Apr;226(1-2):305–309. doi: 10.1007/BF00273617. [DOI] [PubMed] [Google Scholar]
  32. Rossi F., Labourier E., Forné T., Divita G., Derancourt J., Riou J. F., Antoine E., Cathala G., Brunel C., Tazi J. Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature. 1996 May 2;381(6577):80–82. doi: 10.1038/381080a0. [DOI] [PubMed] [Google Scholar]
  33. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  34. Schultz S. J., Nigg E. A. Identification of 21 novel human protein kinases, including 3 members of a family related to the cell cycle regulator nimA of Aspergillus nidulans. Cell Growth Differ. 1993 Oct;4(10):821–830. [PubMed] [Google Scholar]
  35. Shea J. E., Toyn J. H., Johnston L. H. The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression. Nucleic Acids Res. 1994 Dec 25;22(25):5555–5564. doi: 10.1093/nar/22.25.5555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spector D. L. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. doi: 10.1146/annurev.cb.09.110193.001405. [DOI] [PubMed] [Google Scholar]
  37. Strouboulis J., Wolffe A. P. Functional compartmentalization of the nucleus. J Cell Sci. 1996 Aug;109(Pt 8):1991–2000. doi: 10.1242/jcs.109.8.1991. [DOI] [PubMed] [Google Scholar]
  38. Tazi J., Daugeron M. C., Cathala G., Brunel C., Jeanteur P. Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing. J Biol Chem. 1992 Mar 5;267(7):4322–4326. [PubMed] [Google Scholar]
  39. Tazi J., Kornstädt U., Rossi F., Jeanteur P., Cathala G., Brunel C., Lührmann R. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 1993 May 20;363(6426):283–286. doi: 10.1038/363283a0. [DOI] [PubMed] [Google Scholar]
  40. Woppmann A., Will C. L., Kornstädt U., Zuo P., Manley J. L., Lührmann R. Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors. Nucleic Acids Res. 1993 Jun 25;21(12):2815–2822. doi: 10.1093/nar/21.12.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  42. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]
  43. Zuo P., Manley J. L. Functional domains of the human splicing factor ASF/SF2. EMBO J. 1993 Dec;12(12):4727–4737. doi: 10.1002/j.1460-2075.1993.tb06161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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