Skip to main content
PMC home page
RNA logoLink to RNA
. 1999 Jan;5(1):49–65. doi: 10.1017/s1355838299981323

Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: differential sensitivity of introns to mutational inactivation.

C M Romfo 1, S Lakhe-Reddy 1, J A Wise 1
PMCID: PMC1369739  PMID: 9917066

Abstract

The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro. To expand our understanding of how this protein functions in vivo, we have created a null allele of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and employed it in a variety of genetic complementation assays. First, analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations. Second, despite extensive structural conservation, we find that metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59. Third, because the activity of U2AF65 in vitro involves binding to the 3' polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575). Our data indicate that all four introns tested, including two that lack extensive runs of pyrimidines between the branchpoint and 3' splice site, show splicing defects upon shifting to the nonpermissive condition. In all cases, splicing is blocked prior to the first transesterification reaction in the mutants, consistent with the role inferred for human U2AF65 based on in vitro experiments.

Full Text

The Full Text of this article is available as a PDF (661.1 KB).

Selected References

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

  1. 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]
  2. 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]
  3. Althoff S. M., Stevens S. W., Wise J. A. The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe. Mol Cell Biol. 1994 Dec;14(12):7839–7854. doi: 10.1128/mcb.14.12.7839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alvarez C. J., Romfo C. M., Vanhoy R. W., Porter G. L., Wise J. A. Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo. RNA. 1996 May;2(5):404–418. [PMC free article] [PubMed] [Google Scholar]
  5. Basi G., Schmid E., Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene. 1993 Jan 15;123(1):131–136. doi: 10.1016/0378-1119(93)90552-e. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Brennwald P., Liao X., Holm K., Porter G., Wise J. A. Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle. Mol Cell Biol. 1988 Apr;8(4):1580–1590. doi: 10.1128/mcb.8.4.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burd C. G., Dreyfuss G. RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J. 1994 Mar 1;13(5):1197–1204. doi: 10.1002/j.1460-2075.1994.tb06369.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeVoti J., Seydoux G., Beach D., McLeod M. Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO J. 1991 Dec;10(12):3759–3768. doi: 10.1002/j.1460-2075.1991.tb04945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Diamond S. E., Kirkegaard K. Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis. J Virol. 1994 Feb;68(2):863–876. doi: 10.1128/jvi.68.2.863-876.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fleckner J., Zhang M., Valcárcel J., Green M. R. U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev. 1997 Jul 15;11(14):1864–1872. doi: 10.1101/gad.11.14.1864. [DOI] [PubMed] [Google Scholar]
  12. Forsburg S. L. Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 1993 Jun 25;21(12):2955–2956. doi: 10.1093/nar/21.12.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gaur R. K., Valcárcel J., Green M. R. Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein. RNA. 1995 Jun;1(4):407–417. [PMC free article] [PubMed] [Google Scholar]
  14. Gozani O., Potashkin J., Reed R. A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol Cell Biol. 1998 Aug;18(8):4752–4760. doi: 10.1128/mcb.18.8.4752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hindley J., Phear G. A. Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene. 1984 Nov;31(1-3):129–134. doi: 10.1016/0378-1119(84)90203-8. [DOI] [PubMed] [Google Scholar]
  16. Hiraoka Y., Toda T., Yanagida M. The NDA3 gene of fission yeast encodes beta-tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell. 1984 Dec;39(2 Pt 1):349–358. doi: 10.1016/0092-8674(84)90013-8. [DOI] [PubMed] [Google Scholar]
  17. Hoffman B. E., Grabowski P. J. U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon. Genes Dev. 1992 Dec;6(12B):2554–2568. doi: 10.1101/gad.6.12b.2554. [DOI] [PubMed] [Google Scholar]
  18. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [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. Kanaar R., Roche S. E., Beall E. L., Green M. R., Rio D. C. The conserved pre-mRNA splicing factor U2AF from Drosophila: requirement for viability. Science. 1993 Oct 22;262(5133):569–573. doi: 10.1126/science.7692602. [DOI] [PubMed] [Google Scholar]
  21. Kelly M., Burke J., Smith M., Klar A., Beach D. Four mating-type genes control sexual differentiation in the fission yeast. EMBO J. 1988 May;7(5):1537–1547. doi: 10.1002/j.1460-2075.1988.tb02973.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Liao X. B., Brennwald P., Wise J. A. Genetic analysis of Schizosaccharomyces pombe 7SL RNA: a structural motif that includes a conserved tetranucleotide loop is important for function. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4137–4141. doi: 10.1073/pnas.86.11.4137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990 Jul 5;265(19):10857–10864. [PubMed] [Google Scholar]
  25. McKinney R., Wentz-Hunter K., Schmidt H., Potashkin J. Molecular characterization of a novel fission yeast gene spUAP2 that interacts with the splicing factor spU2AF59. Curr Genet. 1997 Nov;32(5):323–330. doi: 10.1007/s002940050284. [DOI] [PubMed] [Google Scholar]
  26. 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]
  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. Moser M. J., Lee S. Y., Klevit R. E., Davis T. N. Ca2+ binding to calmodulin and its role in Schizosaccharomyces pombe as revealed by mutagenesis and NMR spectroscopy. J Biol Chem. 1995 Sep 1;270(35):20643–20652. doi: 10.1074/jbc.270.35.20643. [DOI] [PubMed] [Google Scholar]
  29. Nagai K., Oubridge C., Jessen T. H., Li J., Evans P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature. 1990 Dec 6;348(6301):515–520. doi: 10.1038/348515a0. [DOI] [PubMed] [Google Scholar]
  30. Oubridge C., Ito N., Evans P. R., Teo C. H., Nagai K. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature. 1994 Dec 1;372(6505):432–438. doi: 10.1038/372432a0. [DOI] [PubMed] [Google Scholar]
  31. Patterson B., Guthrie C. A U-rich tract enhances usage of an alternative 3' splice site in yeast. Cell. 1991 Jan 11;64(1):181–187. doi: 10.1016/0092-8674(91)90219-o. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Porter G., Brennwald P., Wise J. A. U1 small nuclear RNA from Schizosaccharomyces pombe has unique and conserved features and is encoded by an essential single-copy gene. Mol Cell Biol. 1990 Jun;10(6):2874–2881. doi: 10.1128/mcb.10.6.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Potashkin J., Frendewey D. Splicing of the U6 RNA precursor is impaired in fission yeast pre-mRNA splicing mutants. Nucleic Acids Res. 1989 Oct 11;17(19):7821–7831. doi: 10.1093/nar/17.19.7821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Potashkin J., Li R., Frendewey D. Pre-mRNA splicing mutants of Schizosaccharomyces pombe. EMBO J. 1989 Feb;8(2):551–559. doi: 10.1002/j.1460-2075.1989.tb03409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Potashkin J., Naik K., Wentz-Hunter K. U2AF homolog required for splicing in vivo. Science. 1993 Oct 22;262(5133):573–575. doi: 10.1126/science.8211184. [DOI] [PubMed] [Google Scholar]
  37. Potashkin J., Naik K., Wentz-Hunter K. U2AF homolog required for splicing in vivo. Science. 1993 Oct 22;262(5133):573–575. doi: 10.1126/science.8211184. [DOI] [PubMed] [Google Scholar]
  38. Rain J. C., Rafi Z., Rhani Z., Legrain P., Krämer A. Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1. RNA. 1998 May;4(5):551–565. doi: 10.1017/s1355838298980335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reich C. I., VanHoy R. W., Porter G. L., Wise J. A. Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast. Cell. 1992 Jun 26;69(7):1159–1169. doi: 10.1016/0092-8674(92)90637-r. [DOI] [PubMed] [Google Scholar]
  40. Reijo R. A., Cooper E. M., Beagle G. J., Huffaker T. C. Systematic mutational analysis of the yeast beta-tubulin gene. Mol Biol Cell. 1994 Jan;5(1):29–43. doi: 10.1091/mbc.5.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Romfo C. M., Wise J. A. Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast. Nucleic Acids Res. 1997 Nov 15;25(22):4658–4665. doi: 10.1093/nar/25.22.4658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rudner D. Z., Breger K. S., Kanaar R., Adams M. D., Rio D. C. RNA binding activity of heterodimeric splicing factor U2AF: at least one RS domain is required for high-affinity binding. Mol Cell Biol. 1998 Jul;18(7):4004–4011. doi: 10.1128/mcb.18.7.4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rudner D. Z., Breger K. S., Rio D. C. Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo. Genes Dev. 1998 Apr 1;12(7):1010–1021. doi: 10.1101/gad.12.7.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  45. Singh R., Valcárcel J., Green M. R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. doi: 10.1126/science.7761834. [DOI] [PubMed] [Google Scholar]
  46. Takahashi K., Yamada H., Yanagida M. Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality. Mol Biol Cell. 1994 Oct;5(10):1145–1158. doi: 10.1091/mbc.5.10.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Takeda T., Yamamoto M. Analysis and in vivo disruption of the gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3580–3584. doi: 10.1073/pnas.84.11.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tani T., Ohshima Y. The gene for the U6 small nuclear RNA in fission yeast has an intron. Nature. 1989 Jan 5;337(6202):87–90. doi: 10.1038/337087a0. [DOI] [PubMed] [Google Scholar]
  49. Urushiyama S., Tani T., Ohshima Y. Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe. Mol Gen Genet. 1996 Nov 27;253(1-2):118–127. doi: 10.1007/s004380050304. [DOI] [PubMed] [Google Scholar]
  50. Valcárcel J., Gaur R. K., Singh R., Green M. R. Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected]. Science. 1996 Sep 20;273(5282):1706–1709. doi: 10.1126/science.273.5282.1706. [DOI] [PubMed] [Google Scholar]
  51. Valcárcel J., Singh R., Zamore P. D., Green M. R. The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature. 1993 Mar 11;362(6416):171–175. doi: 10.1038/362171a0. [DOI] [PubMed] [Google Scholar]
  52. Wentz-Hunter K., Potashkin J. The small subunit of the splicing factor U2AF is conserved in fission yeast. Nucleic Acids Res. 1996 May 15;24(10):1849–1854. doi: 10.1093/nar/24.10.1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zamore P. D., Green M. R. Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 1991 Jan;10(1):207–214. doi: 10.1002/j.1460-2075.1991.tb07937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zamore P. D., Green M. R. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]
  57. Zhang M. Q., Marr T. G. Fission yeast gene structure and recognition. Nucleic Acids Res. 1994 May 11;22(9):1750–1759. doi: 10.1093/nar/22.9.1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhang M., Zamore P. D., Carmo-Fonseca M., Lamond A. I., Green M. R. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8769–8773. doi: 10.1073/pnas.89.18.8769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. Zorio D. A., Lea K., Blumenthal T. Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon. Mol Cell Biol. 1997 Feb;17(2):946–953. doi: 10.1128/mcb.17.2.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zuo P., Maniatis T. The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev. 1996 Jun 1;10(11):1356–1368. doi: 10.1101/gad.10.11.1356. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES