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Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Mar;16(3):960–967. doi: 10.1128/mcb.16.3.960

Identification of Prp40, a novel essential yeast splicing factor associated with the U1 small nuclear ribonucleoprotein particle.

H Y Kao 1, P G Siliciano 1
PMCID: PMC231078  PMID: 8622699

Abstract

We have used suppressor genetics to identify factors that interact with Saccharomyces cerevisiae U1 small nuclear RNA (snRNA). In this way, we isolated PRP40-1, a suppressor that restores growth at 18 degrees C to a strain bearing a cold-sensitive mutation in U1 RNA. A gene disruption experiment shows that PRP40 is an essential gene. To study the role of PRP40 in splicing, we created a pool of temperature-sensitive prp40 strains. Primer extension analysis of intron-containing transcripts in prp40 temperature-sensitive strains reveals a splicing defect, indicating that Prp40 plays a direct role in pre-mRNA splicing. In addition, U1 RNA coimmunoprecipitates with Pro40, indicating that Prp40 is bound to the U1 small nuclear ribonucleoprotein particle in vivo. Therefore, we conclude that PRP40 encodes a novel, essential splicing component that associates with the yeast U1 small nuclear ribonucleoprotein particle.

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Selected References

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  1. André B., Springael J. Y. WWP, a new amino acid motif present in single or multiple copies in various proteins including dystrophin and the SH3-binding Yes-associated protein YAP65. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1201–1205. doi: 10.1006/bbrc.1994.2793. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bork P., Sudol M. The WW domain: a signalling site in dystrophin? Trends Biochem Sci. 1994 Dec;19(12):531–533. doi: 10.1016/0968-0004(94)90053-1. [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. Cheng S. C., Abelson J. Fractionation and characterization of a yeast mRNA splicing extract. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2387–2391. doi: 10.1073/pnas.83.8.2387. [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. Dabeva M. D., Post-Beittenmiller M. A., Warner J. R. Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5854–5857. doi: 10.1073/pnas.83.16.5854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dabeva M. D., Warner J. R. The yeast ribosomal protein L32 and its gene. J Biol Chem. 1987 Nov 25;262(33):16055–16059. [PubMed] [Google Scholar]
  9. 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]
  10. Elledge S. J., Davis R. W. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. doi: 10.1016/0378-1119(88)90202-8. [DOI] [PubMed] [Google Scholar]
  11. 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]
  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. 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]
  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. Hamm J., van Santen V. L., Spritz R. A., Mattaj I. W. Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins. Mol Cell Biol. 1988 Nov;8(11):4787–4791. doi: 10.1128/mcb.8.11.4787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. He F., Peltz S. W., Donahue J. L., Rosbash M., Jacobson A. Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7034–7038. doi: 10.1073/pnas.90.15.7034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hilleren P. J., Kao H. Y., Siliciano P. G. The amino-terminal domain of yeast U1-70K is necessary and sufficient for function. Mol Cell Biol. 1995 Nov;15(11):6341–6350. doi: 10.1128/mcb.15.11.6341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnston G. C., Prendergast J. A., Singer R. A. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. doi: 10.1083/jcb.113.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones M. H., Guthrie C. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 1990 Aug;9(8):2555–2561. doi: 10.1002/j.1460-2075.1990.tb07436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kao H. Y., Siliciano P. G. The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene. Nucleic Acids Res. 1992 Aug 11;20(15):4009–4013. doi: 10.1093/nar/20.15.4009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kretzner L., Krol A., Rosbash M. Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains. Proc Natl Acad Sci U S A. 1990 Jan;87(2):851–855. doi: 10.1073/pnas.87.2.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Legrain P., Seraphin B., Rosbash M. Early commitment of yeast pre-mRNA to the spliceosome pathway. Mol Cell Biol. 1988 Sep;8(9):3755–3760. doi: 10.1128/mcb.8.9.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liao X. C., Colot H. V., Wang Y., Rosbash M. Requirements for U2 snRNP addition to yeast pre-mRNA. Nucleic Acids Res. 1992 Aug 25;20(16):4237–4245. doi: 10.1093/nar/20.16.4237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Lutz-Freyermuth C., Keene J. D., Lutz-Reyermuth C. The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs. Mol Cell Biol. 1989 Jul;9(7):2975–2982. doi: 10.1128/mcb.9.7.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Michaud S., Reed R. A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals. Genes Dev. 1993 Jun;7(6):1008–1020. doi: 10.1101/gad.7.6.1008. [DOI] [PubMed] [Google Scholar]
  30. Michaud S., Reed R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991 Dec;5(12B):2534–2546. doi: 10.1101/gad.5.12b.2534. [DOI] [PubMed] [Google Scholar]
  31. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  32. Mount S. M., Steitz J. A. Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing. Nucleic Acids Res. 1981 Dec 11;9(23):6351–6368. doi: 10.1093/nar/9.23.6351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nakańo A., Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nelissen R. L., Will C. L., van Venrooij W. J., Lührmann R. The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins. EMBO J. 1994 Sep 1;13(17):4113–4125. doi: 10.1002/j.1460-2075.1994.tb06729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pascolo S., Ghazvini M., Boyer J., Colleaux L., Thierry A., Dujon B. The sequence of a 9.3 kb segment located on the left arm of the yeast chromosome XI reveals five open reading frames including the CCE1 gene and putative products related to MYO2 and to the ribosomal protein L10. Yeast. 1992 Nov;8(11):987–995. doi: 10.1002/yea.320081109. [DOI] [PubMed] [Google Scholar]
  36. Pikielny C. W., Rosbash M. mRNA splicing efficiency in yeast and the contribution of nonconserved sequences. Cell. 1985 May;41(1):119–126. doi: 10.1016/0092-8674(85)90066-2. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  39. Rose M. D., Broach J. R. Cloning genes by complementation in yeast. Methods Enzymol. 1991;194:195–230. doi: 10.1016/0076-6879(91)94017-7. [DOI] [PubMed] [Google Scholar]
  40. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  41. Schena M., Picard D., Yamamoto K. R. Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol. 1991;194:389–398. doi: 10.1016/0076-6879(91)94029-c. [DOI] [PubMed] [Google Scholar]
  42. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Siliciano P. G., Brow D. A., Roiha H., Guthrie C. An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA. Cell. 1987 Aug 14;50(4):585–592. doi: 10.1016/0092-8674(87)90031-6. [DOI] [PubMed] [Google Scholar]
  46. Siliciano P. G., Guthrie C. 5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev. 1988 Oct;2(10):1258–1267. doi: 10.1101/gad.2.10.1258. [DOI] [PubMed] [Google Scholar]
  47. Smith V., Barrell B. G. Cloning of a yeast U1 snRNP 70K protein homologue: functional conservation of an RNA-binding domain between humans and yeast. EMBO J. 1991 Sep;10(9):2627–2634. doi: 10.1002/j.1460-2075.1991.tb07805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sudol M., Bork P., Einbond A., Kastury K., Druck T., Negrini M., Huebner K., Lehman D. Characterization of the mammalian YAP (Yes-associated protein) gene and its role in defining a novel protein module, the WW domain. J Biol Chem. 1995 Jun 16;270(24):14733–14741. doi: 10.1074/jbc.270.24.14733. [DOI] [PubMed] [Google Scholar]
  49. Surowy C. S., van Santen V. L., Scheib-Wixted S. M., Spritz R. A. Direct, sequence-specific binding of the human U1-70K ribonucleoprotein antigen protein to loop I of U1 small nuclear RNA. Mol Cell Biol. 1989 Oct;9(10):4179–4186. doi: 10.1128/mcb.9.10.4179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Séraphin B., Kretzner L., Rosbash M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site. EMBO J. 1988 Aug;7(8):2533–2538. doi: 10.1002/j.1460-2075.1988.tb03101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Séraphin B., Rosbash M. The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA. EMBO J. 1991 May;10(5):1209–1216. doi: 10.1002/j.1460-2075.1991.tb08062.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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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