Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jul;17(7):3580–3588. doi: 10.1128/mcb.17.7.3580

Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae.

E M Weidenhammer 1, M Ruiz-Noriega 1, J L Woolford Jr 1
PMCID: PMC232211  PMID: 9199293

Abstract

The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Abovich N., Legrain P., Rosbash M. The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding. Mol Cell Biol. 1990 Dec;10(12):6417–6425. doi: 10.1128/mcb.10.12.6417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Adams M. D., Rudner D. Z., Rio D. C. Biochemistry and regulation of pre-mRNA splicing. Curr Opin Cell Biol. 1996 Jun;8(3):331–339. doi: 10.1016/s0955-0674(96)80006-8. [DOI] [PubMed] [Google Scholar]
  4. Arenas J. E., Abelson J. N. The Saccharomyces cerevisiae PRP21 gene product is an integral component of the prespliceosome. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6771–6775. doi: 10.1073/pnas.90.14.6771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ares M., Jr, Weiser B. Rearrangement of snRNA structure during assembly and function of the spliceosome. Prog Nucleic Acid Res Mol Biol. 1995;50:131–159. doi: 10.1016/s0079-6603(08)60813-2. [DOI] [PubMed] [Google Scholar]
  6. Banroques J., Abelson J. N. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3710–3719. doi: 10.1128/mcb.9.9.3710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Behrens S. E., Lührmann R. Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells. Genes Dev. 1991 Aug;5(8):1439–1452. doi: 10.1101/gad.5.8.1439. [DOI] [PubMed] [Google Scholar]
  8. Bindereif A., Green M. R. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. doi: 10.1002/j.1460-2075.1987.tb02520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bjørn S. P., Soltyk A., Beggs J. D., Friesen J. D. PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3698–3709. doi: 10.1128/mcb.9.9.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Black D. L., Pinto A. L. U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6. Mol Cell Biol. 1989 Aug;9(8):3350–3359. doi: 10.1128/mcb.9.8.3350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Blanton S., Srinivasan A., Rymond B. C. PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels. Mol Cell Biol. 1992 Sep;12(9):3939–3947. doi: 10.1128/mcb.12.9.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bordonné R., Banroques J., Abelson J., Guthrie C. Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo. Genes Dev. 1990 Jul;4(7):1185–1196. doi: 10.1101/gad.4.7.1185. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Brow D. A., Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988 Jul 21;334(6179):213–218. doi: 10.1038/334213a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Fabrizio P., Abelson J. Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing. Science. 1990 Oct 19;250(4979):404–409. doi: 10.1126/science.2145630. [DOI] [PubMed] [Google Scholar]
  18. Fabrizio P., Esser S., Kastner B., Lührmann R. Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts. Science. 1994 Apr 8;264(5156):261–265. doi: 10.1126/science.8146658. [DOI] [PubMed] [Google Scholar]
  19. Fabrizio P., McPheeters D. S., Abelson J. In vitro assembly of yeast U6 snRNP: a functional assay. Genes Dev. 1989 Dec;3(12B):2137–2150. doi: 10.1101/gad.3.12b.2137. [DOI] [PubMed] [Google Scholar]
  20. Fortner D. M., Troy R. G., Brow D. A. A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing. Genes Dev. 1994 Jan;8(2):221–233. doi: 10.1101/gad.8.2.221. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  23. Galisson F., Legrain P. The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. Nucleic Acids Res. 1993 Apr 11;21(7):1555–1562. doi: 10.1093/nar/21.7.1555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Guthrie C. The spliceosome is a dynamic ribonucleoprotein machine. Harvey Lect. 1994;90:59–80. [PubMed] [Google Scholar]
  27. Hodges P. E., Beggs J. D. RNA splicing. U2 fulfils a commitment. Curr Biol. 1994 Mar 1;4(3):264–267. doi: 10.1016/s0960-9822(00)00061-0. [DOI] [PubMed] [Google Scholar]
  28. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  29. Lamm G. M., Blencowe B. J., Sproat B. S., Iribarren A. M., Ryder U., Lamond A. I. Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts. Nucleic Acids Res. 1991 Jun 25;19(12):3193–3198. doi: 10.1093/nar/19.12.3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li Z., Brow D. A. A rapid assay for quantitative detection of specific RNAs. Nucleic Acids Res. 1993 Sep 25;21(19):4645–4646. doi: 10.1093/nar/21.19.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liao X. C., Tang J., Rosbash M. An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing. Genes Dev. 1993 Mar;7(3):419–428. doi: 10.1101/gad.7.3.419. [DOI] [PubMed] [Google Scholar]
  32. Lin R. J., Newman A. J., Cheng S. C., Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
  33. Lockhart S. R., Rymond B. C. Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p. Mol Cell Biol. 1994 Jun;14(6):3623–3633. doi: 10.1128/mcb.14.6.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  36. Maddock J. R., Roy J., Woolford J. L., Jr Six novel genes necessary for pre-mRNA splicing in Saccharomyces cerevisiae. Nucleic Acids Res. 1996 Mar 15;24(6):1037–1044. doi: 10.1093/nar/24.6.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. Nilsen T. W. RNA-RNA interactions in the spliceosome: unraveling the ties that bind. Cell. 1994 Jul 15;78(1):1–4. doi: 10.1016/0092-8674(94)90563-0. [DOI] [PubMed] [Google Scholar]
  41. Pikielny C. W., Rosbash M. Specific small nuclear RNAs are associated with yeast spliceosomes. Cell. 1986 Jun 20;45(6):869–877. doi: 10.1016/0092-8674(86)90561-1. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Rosbash M., Séraphin B. Who's on first? The U1 snRNP-5' splice site interaction and splicing. Trends Biochem Sci. 1991 May;16(5):187–190. doi: 10.1016/0968-0004(91)90073-5. [DOI] [PubMed] [Google Scholar]
  45. Roscigno R. F., Garcia-Blanco M. A. SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome. RNA. 1995 Sep;1(7):692–706. [PMC free article] [PubMed] [Google Scholar]
  46. Roy J., Zheng B., Rymond B. C., Woolford J. L., Jr Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins. Mol Cell Biol. 1995 Jan;15(1):445–455. doi: 10.1128/mcb.15.1.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ruby S. W., Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. doi: 10.1016/0168-9525(91)90276-V. [DOI] [PubMed] [Google Scholar]
  48. Rymond B. C., Rosbash M. Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing. Nature. 1985 Oct 24;317(6039):735–737. doi: 10.1038/317735a0. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Shannon K. W., Guthrie C. Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs. Genes Dev. 1991 May;5(5):773–785. doi: 10.1101/gad.5.5.773. [DOI] [PubMed] [Google Scholar]
  51. Séraphin B., Abovich N., Rosbash M. Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly. Nucleic Acids Res. 1991 Jul 25;19(14):3857–3860. doi: 10.1093/nar/19.14.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Utans U., Behrens S. E., Lührmann R., Kole R., Krämer A. A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins. Genes Dev. 1992 Apr;6(4):631–641. doi: 10.1101/gad.6.4.631. [DOI] [PubMed] [Google Scholar]
  53. Valcárcel J., Green M. R. The SR protein family: pleiotropic functions in pre-mRNA splicing. Trends Biochem Sci. 1996 Aug;21(8):296–301. [PubMed] [Google Scholar]
  54. Weidenhammer E. M., Singh M., Ruiz-Noriega M., Woolford J. L., Jr The PRP31 gene encodes a novel protein required for pre-mRNA splicing in Saccharomyces cerevisiae. Nucleic Acids Res. 1996 Mar 15;24(6):1164–1170. doi: 10.1093/nar/24.6.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xu Y., Petersen-Bjørn S., Friesen J. D. The PRP4 (RNA4) protein of Saccharomyces cerevisiae is associated with the 5' portion of the U4 small nuclear RNA. Mol Cell Biol. 1990 Mar;10(3):1217–1225. doi: 10.1128/mcb.10.3.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zagorski J., Tollervey D., Fournier M. J. Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs. Mol Cell Biol. 1988 Aug;8(8):3282–3290. doi: 10.1128/mcb.8.8.3282. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES