Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae (original) (raw)

Abstract

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.

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Selected References

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  1. Abovich N., Rosbash M. Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell. 1997 May 2;89(3):403–412. doi: 10.1016/s0092-8674(00)80221-4. [DOI] [PubMed] [Google Scholar]
  2. Achsel T., Ahrens K., Brahms H., Teigelkamp S., Lührmann R. The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein. Mol Cell Biol. 1998 Nov;18(11):6756–6766. doi: 10.1128/mcb.18.11.6756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ben-Yehuda S., Russell C. S., Dix I., Beggs J. D., Kupiec M. Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression. Genetics. 2000 Jan;154(1):61–71. doi: 10.1093/genetics/154.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boronenkov I. V., Loijens J. C., Umeda M., Anderson R. A. Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol Biol Cell. 1998 Dec;9(12):3547–3560. doi: 10.1091/mbc.9.12.3547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown J. D., Beggs J. D. Roles of PRP8 protein in the assembly of splicing complexes. EMBO J. 1992 Oct;11(10):3721–3729. doi: 10.1002/j.1460-2075.1992.tb05457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chang T. H., Latus L. J., Liu Z., Abbott J. M. Genetic interactions of conserved regions in the DEAD-box protein Prp28p. Nucleic Acids Res. 1997 Dec 15;25(24):5033–5040. doi: 10.1093/nar/25.24.5033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chong J. P., Hayashi M. K., Simon M. N., Xu R. M., Stillman B. A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1530–1535. doi: 10.1073/pnas.030539597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collins C. A., Guthrie C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 1999 Aug 1;13(15):1970–1982. doi: 10.1101/gad.13.15.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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 Dec;4(12):1675–1686. doi: 10.1017/s1355838298412998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fabrizio P., Laggerbauer B., Lauber J., Lane W. S., Lührmann R. An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2. EMBO J. 1997 Jul 1;16(13):4092–4106. doi: 10.1093/emboj/16.13.4092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fromont-Racine M., Mayes A. E., Brunet-Simon A., Rain J. C., Colley A., Dix I., Decourty L., Joly N., Ricard F., Beggs J. D. Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins. Yeast. 2000 Jun 30;17(2):95–110. doi: 10.1002/1097-0061(20000630)17:2<95::AID-YEA16>3.0.CO;2-H. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fromont-Racine M., Rain J. C., Legrain P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat Genet. 1997 Jul;16(3):277–282. doi: 10.1038/ng0797-277. [DOI] [PubMed] [Google Scholar]
  14. Gottschalk A., Neubauer G., Banroques J., Mann M., Lührmann R., Fabrizio P. Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP. EMBO J. 1999 Aug 16;18(16):4535–4548. doi: 10.1093/emboj/18.16.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Ishikawa S., Kai M., Tamari M., Takei Y., Takeuchi K., Bandou H., Yamane Y., Ogawa M., Nakamura Y. Sequence analysis of a 685-kb genomic region on chromosome 3p22-p21.3 that is homozygously deleted in a lung carcinoma cell line. DNA Res. 1997 Feb 28;4(1):35–43. doi: 10.1093/dnares/4.1.35. [DOI] [PubMed] [Google Scholar]
  17. Jackson S. P., Lossky M., Beggs J. D. Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing. Mol Cell Biol. 1988 Mar;8(3):1067–1075. doi: 10.1128/mcb.8.3.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim D. H., Rossi J. J. The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex. RNA. 1999 Jul;5(7):959–971. doi: 10.1017/s135583829999012x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim S. H., Lin R. J. Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol Cell Biol. 1996 Dec;16(12):6810–6819. doi: 10.1128/mcb.16.12.6810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuhn A. N., Brow D. A. Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation. Genetics. 2000 Aug;155(4):1667–1682. doi: 10.1093/genetics/155.4.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lauber J., Fabrizio P., Teigelkamp S., Lane W. S., Hartmann E., Luhrmann R. The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases. EMBO J. 1996 Aug 1;15(15):4001–4015. [PMC free article] [PubMed] [Google Scholar]
  22. Lee J. K., Hurwitz J. Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe. J Biol Chem. 2000 Jun 23;275(25):18871–18878. doi: 10.1074/jbc.M001118200. [DOI] [PubMed] [Google Scholar]
  23. Levin M. K., Patel S. S. The helicase from hepatitis C virus is active as an oligomer. J Biol Chem. 1999 Nov 5;274(45):31839–31846. doi: 10.1074/jbc.274.45.31839. [DOI] [PubMed] [Google Scholar]
  24. Mayes A. E., Verdone L., Legrain P., Beggs J. D. Characterization of Sm-like proteins in yeast and their association with U6 snRNA. EMBO J. 1999 Aug 2;18(15):4321–4331. doi: 10.1093/emboj/18.15.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McPheeters D. S., Schwer B., Muhlenkamp P. Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing. Nucleic Acids Res. 2000 Mar 15;28(6):1313–1321. doi: 10.1093/nar/28.6.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mortillaro M. J., Berezney R. Matrin CYP, an SR-rich cyclophilin that associates with the nuclear matrix and splicing factors. J Biol Chem. 1998 Apr 3;273(14):8183–8192. doi: 10.1074/jbc.273.14.8183. [DOI] [PubMed] [Google Scholar]
  27. Newman A. J., Norman C. U5 snRNA interacts with exon sequences at 5' and 3' splice sites. Cell. 1992 Feb 21;68(4):743–754. doi: 10.1016/0092-8674(92)90149-7. [DOI] [PubMed] [Google Scholar]
  28. Newman A. J., Teigelkamp S., Beggs J. D. snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes. RNA. 1995 Nov;1(9):968–980. [PMC free article] [PubMed] [Google Scholar]
  29. O'Keefe R. T., Newman A. J. Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing. EMBO J. 1998 Jan 15;17(2):565–574. doi: 10.1093/emboj/17.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Raghunathan P. L., Guthrie C. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol. 1998 Jul 16;8(15):847–855. doi: 10.1016/s0960-9822(07)00345-4. [DOI] [PubMed] [Google Scholar]
  31. Rain J. C., Legrain P. In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein. EMBO J. 1997 Apr 1;16(7):1759–1771. doi: 10.1093/emboj/16.7.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reyes J. L., Gustafson E. H., Luo H. R., Moore M. J., Konarska M. M. The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site. RNA. 1999 Feb;5(2):167–179. doi: 10.1017/s1355838299981785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saiz J. E., Santos M. A., Vázquez de Aldana C. R., Revuelta J. L. Disruption of six unknown open reading frames from Saccharomyces cerevisiae reveals two genes involved in vacuolar morphogenesis and one gene required for sporulation. Yeast. 1999 Jan 30;15(2):155–164. doi: 10.1002/(SICI)1097-0061(19990130)15:2<155::AID-YEA342>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  34. Schwer B., Gross C. H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 1998 Apr 1;17(7):2086–2094. doi: 10.1093/emboj/17.7.2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Staley J. P., Guthrie C. An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p. Mol Cell. 1999 Jan;3(1):55–64. doi: 10.1016/s1097-2765(00)80174-4. [DOI] [PubMed] [Google Scholar]
  36. Stevens S. W., Abelson J. Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7226–7231. doi: 10.1073/pnas.96.13.7226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Su Y. A., Lee M. M., Hutter C. M., Meltzer P. S. Characterization of a highly conserved gene (OS4) amplified with CDK4 in human sarcomas. Oncogene. 1997 Sep;15(11):1289–1294. doi: 10.1038/sj.onc.1201294. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Vidal V. P., Verdone L., Mayes A. E., Beggs J. D. Characterization of U6 snRNA-protein interactions. RNA. 1999 Nov;5(11):1470–1481. doi: 10.1017/s1355838299991355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wagner J. D., Jankowsky E., Company M., Pyle A. M., Abelson J. N. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes. EMBO J. 1998 May 15;17(10):2926–2937. doi: 10.1093/emboj/17.10.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang Y., Guthrie C. PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain. RNA. 1998 Oct;4(10):1216–1229. [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. West S. C. DNA helicases: new breeds of translocating motors and molecular pumps. Cell. 1996 Jul 26;86(2):177–180. doi: 10.1016/s0092-8674(00)80088-4. [DOI] [PubMed] [Google Scholar]
  46. Wishart M. J., Dixon J. E. Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem Sci. 1998 Aug;23(8):301–306. doi: 10.1016/s0968-0004(98)01241-9. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Xu D., Nouraini S., Field D., Tang S. J., Friesen J. D. An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing. Nature. 1996 Jun 20;381(6584):709–713. doi: 10.1038/381709a0. [DOI] [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]