Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2000 Jan;154(1):61–71. doi: 10.1093/genetics/154.1.61

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression.

S Ben-Yehuda 1, C S Russell 1, I Dix 1, J D Beggs 1, M Kupiec 1
PMCID: PMC1460917  PMID: 10628969

Abstract

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.

Full Text

The Full Text of this article is available as a PDF (216.1 KB).

Selected References

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

  1. Anderson G. J., Bach M., Lührmann R., Beggs J. D. Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein. Nature. 1989 Dec 14;342(6251):819–821. doi: 10.1038/342819a0. [DOI] [PubMed] [Google Scholar]
  2. Ben Yehuda S., Dix I., Russell C. S., Levy S., Beggs J. D., Kupiec M. Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control. RNA. 1998 Oct;4(10):1304–1312. doi: 10.1017/s1355838298980712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  4. Boger-Nadjar E., Vaisman N., Ben-Yehuda S., Kassir Y., Kupiec M. Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing. Mol Gen Genet. 1998 Nov;260(2-3):232–241. doi: 10.1007/s004380050891. [DOI] [PubMed] [Google Scholar]
  5. Bone R., Fujishige A., Kettner C. A., Agard D. A. Structural basis for broad specificity in alpha-lytic protease mutants. Biochemistry. 1991 Oct 29;30(43):10388–10398. doi: 10.1021/bi00107a005. [DOI] [PubMed] [Google Scholar]
  6. Boorstein W. R., Craig E. A. Primer extension analysis of RNA. Methods Enzymol. 1989;180:347–369. doi: 10.1016/0076-6879(89)80111-9. [DOI] [PubMed] [Google Scholar]
  7. Brys A., Schwer B. Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site. RNA. 1996 Jul;2(7):707–717. [PMC free article] [PubMed] [Google Scholar]
  8. Burgess S. M., Guthrie C. A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell. 1993 Jul 2;73(7):1377–1391. doi: 10.1016/0092-8674(93)90363-u. [DOI] [PubMed] [Google Scholar]
  9. Dix I., Russell C. S., O'Keefe R. T., Newman A. J., Beggs J. D. Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. RNA. 1998 Oct;4(10):1239–1250. doi: 10.1017/s1355838298981109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dumas L. B., Lussky J. P., McFarland E. J., Shampay J. New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication. Mol Gen Genet. 1982;187(1):42–46. doi: 10.1007/BF00384381. [DOI] [PubMed] [Google Scholar]
  11. Elledge S. J. Cell cycle checkpoints: preventing an identity crisis. Science. 1996 Dec 6;274(5293):1664–1672. doi: 10.1126/science.274.5293.1664. [DOI] [PubMed] [Google Scholar]
  12. Frank D., Guthrie C. An essential splicing factor, SLU7, mediates 3' splice site choice in yeast. Genes Dev. 1992 Nov;6(11):2112–2124. doi: 10.1101/gad.6.11.2112. [DOI] [PubMed] [Google Scholar]
  13. Frank D., Patterson B., Guthrie C. Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing. Mol Cell Biol. 1992 Nov;12(11):5197–5205. doi: 10.1128/mcb.12.11.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  15. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  16. Hodges P. E., Jackson S. P., Brown J. D., Beggs J. D. Extraordinary sequence conservation of the PRP8 splicing factor. Yeast. 1995 Apr 15;11(4):337–342. doi: 10.1002/yea.320110406. [DOI] [PubMed] [Google Scholar]
  17. Horowitz D. S., Abelson J. Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent. Genes Dev. 1993 Feb;7(2):320–329. doi: 10.1101/gad.7.2.320. [DOI] [PubMed] [Google Scholar]
  18. Hotz H. R., Schwer B. Mutational analysis of the yeast DEAH-box splicing factor Prp16. Genetics. 1998 Jun;149(2):807–815. doi: 10.1093/genetics/149.2.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones M. H., Frank D. N., Guthrie C. Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9687–9691. doi: 10.1073/pnas.92.21.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kassir Y., Kupiec M., Shalom A., Simchen G. Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair. Curr Genet. 1985;9(4):253–257. doi: 10.1007/BF00419952. [DOI] [PubMed] [Google Scholar]
  21. Krämer A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem. 1996;65:367–409. doi: 10.1146/annurev.bi.65.070196.002055. [DOI] [PubMed] [Google Scholar]
  22. Kuhn A. N., Li Z., Brow D. A. Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome. Mol Cell. 1999 Jan;3(1):65–75. doi: 10.1016/s1097-2765(00)80175-6. [DOI] [PubMed] [Google Scholar]
  23. Lesser C. F., Guthrie C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science. 1993 Dec 24;262(5142):1982–1988. doi: 10.1126/science.8266093. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Madhani H. D., Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet. 1994;28:1–26. doi: 10.1146/annurev.ge.28.120194.000245. [DOI] [PubMed] [Google Scholar]
  26. Neer E. J., Schmidt C. J., Nambudripad R., Smith T. F. The ancient regulatory-protein family of WD-repeat proteins. Nature. 1994 Sep 22;371(6495):297–300. doi: 10.1038/371297a0. [DOI] [PubMed] [Google Scholar]
  27. Opresko P. L., Sweasy J. B., Eckert K. A. The mutator form of polymerase beta with amino acid substitution at tyrosine 265 in the hinge region displays an increase in both base substitution and frame shift errors. Biochemistry. 1998 Feb 24;37(8):2111–2119. doi: 10.1021/bi9722711. [DOI] [PubMed] [Google Scholar]
  28. Pelletier H., Sawaya M. R., Wolfle W., Wilson S. H., Kraut J. Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity. Biochemistry. 1996 Oct 1;35(39):12742–12761. doi: 10.1021/bi952955d. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [DOI] [PubMed] [Google Scholar]
  31. Seghezzi W., Chua K., Shanahan F., Gozani O., Reed R., Lees E. Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells. Mol Cell Biol. 1998 Aug;18(8):4526–4536. doi: 10.1128/mcb.18.8.4526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seshadri V., Vaidya V. C., Vijayraghavan U. Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein. Genetics. 1996 May;143(1):45–55. doi: 10.1093/genetics/143.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Sikorski R. S., Boguski M. S., Goebl M., Hieter P. A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell. 1990 Jan 26;60(2):307–317. doi: 10.1016/0092-8674(90)90745-z. [DOI] [PubMed] [Google Scholar]
  35. Teigelkamp S., Newman A. J., Beggs J. D. Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA. EMBO J. 1995 Jun 1;14(11):2602–2612. doi: 10.1002/j.1460-2075.1995.tb07258.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Teigelkamp S., Whittaker E., Beggs J. D. Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing. Nucleic Acids Res. 1995 Feb 11;23(3):320–326. doi: 10.1093/nar/23.3.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Umen J. G., Guthrie C. Mutagenesis of the yeast gene PRP8 reveals domains governing the specificity and fidelity of 3' splice site selection. Genetics. 1996 Jun;143(2):723–739. doi: 10.1093/genetics/143.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Umen J. G., Guthrie C. Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA. 1995 Aug;1(6):584–597. [PMC free article] [PubMed] [Google Scholar]
  40. Umen J. G., Guthrie C. The second catalytic step of pre-mRNA splicing. RNA. 1995 Nov;1(9):869–885. [PMC free article] [PubMed] [Google Scholar]
  41. Vaisman N., Tsouladze A., Robzyk K., Ben-Yehuda S., Kupiec M., Kassir Y. The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance. Mol Gen Genet. 1995 Apr 20;247(2):123–136. doi: 10.1007/BF00705642. [DOI] [PubMed] [Google Scholar]
  42. Wang J., Manley J. L. Regulation of pre-mRNA splicing in metazoa. Curr Opin Genet Dev. 1997 Apr;7(2):205–211. doi: 10.1016/s0959-437x(97)80130-x. [DOI] [PubMed] [Google Scholar]
  43. Wang Y., Wagner J. D., Guthrie C. The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro. Curr Biol. 1998 Apr 9;8(8):441–451. doi: 10.1016/s0960-9822(98)70178-2. [DOI] [PubMed] [Google Scholar]
  44. Whittaker E., Lossky M., Beggs J. D. Affinity purification of spliceosomes reveals that the precursor RNA processing protein PRP8, a protein in the U5 small nuclear ribonucleoprotein particle, is a component of yeast spliceosomes. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2216–2219. doi: 10.1073/pnas.87.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Will C. L., Lührmann R. Protein functions in pre-mRNA splicing. Curr Opin Cell Biol. 1997 Jun;9(3):320–328. doi: 10.1016/s0955-0674(97)80003-8. [DOI] [PubMed] [Google Scholar]
  46. Williams F. E., Varanasi U., Trumbly R. J. The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex. Mol Cell Biol. 1991 Jun;11(6):3307–3316. doi: 10.1128/mcb.11.6.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Woolford J. L., Jr, Peebles C. L. RNA splicing in lower eukaryotes. Curr Opin Genet Dev. 1992 Oct;2(5):712–719. doi: 10.1016/s0959-437x(05)80131-5. [DOI] [PubMed] [Google Scholar]
  48. Xu D., Field D. J., Tang S. J., Moris A., Bobechko B. P., Friesen J. D. Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing. Mol Cell Biol. 1998 Apr;18(4):2055–2066. doi: 10.1128/mcb.18.4.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhang X., Schwer B. Functional and physical interaction between the yeast splicing factors Slu7 and Prp18. Nucleic Acids Res. 1997 Jun 1;25(11):2146–2152. doi: 10.1093/nar/25.11.2146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhou Z., Reed R. Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing. EMBO J. 1998 Apr 1;17(7):2095–2106. doi: 10.1093/emboj/17.7.2095. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES