Skip to main content
PMC home page
RNA logoLink to RNA
. 1998 Jan;4(1):1–10.

Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits.

H Igel 1, S Wells 1, R Perriman 1, M Ares Jr 1
PMCID: PMC1369591  PMID: 9436903

Abstract

Human SAP 49, a subunit of the multimeric splicing factor 3b (SF3b), contains two RNA recognition motifs (RRMs) and binds another SF3b subunit called SAP 145, whose yeast homologue is CUS1. Here we show that the predicted yeast open reading frame YOR319w (HSH49) encodes an essential yeast splicing factor. Using bacterially expressed proteins, we find that yeast HSH49 binds CUS1. Mutations that alter putative RNA-binding residues of either HSH49 RRM are lethal in vivo, but do not prevent binding to CUS1 in vitro, suggesting that the predicted RNA-binding surfaces of HSH49 are not required for interaction with CUS1. In vivo interaction tests show that HSH49 and CUS1 associate primarily through the N-terminal RRM of HSH49. Recombinant HSH49 protein has a general RNA-binding activity that does not require CUS1. The parallels in structure and interaction between two SF3b subunits from yeast implies that the mechanism of SF3b action is highly conserved.

Full Text

The Full Text of this article is available as a PDF (376.7 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Behrens S. E., Tyc K., Kastner B., Reichelt J., Lührmann R. Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol Cell Biol. 1993 Jan;13(1):307–319. doi: 10.1128/mcb.13.1.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett M., Michaud S., Kingston J., Reed R. Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev. 1992 Oct;6(10):1986–2000. doi: 10.1101/gad.6.10.1986. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Brosi R., Gröning K., Behrens S. E., Lührmann R., Krämer A. Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9. Science. 1993 Oct 1;262(5130):102–105. doi: 10.1126/science.8211112. [DOI] [PubMed] [Google Scholar]
  8. Champion-Arnaud P., Reed R. The prespliceosome components SAP 49 and SAP 145 interact in a complex implicated in tethering U2 snRNP to the branch site. Genes Dev. 1994 Aug 15;8(16):1974–1983. doi: 10.1101/gad.8.16.1974. [DOI] [PubMed] [Google Scholar]
  9. Crowe J., Döbeli H., Gentz R., Hochuli E., Stüber D., Henco K. 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Methods Mol Biol. 1994;31:371–387. doi: 10.1385/0-89603-258-2:371. [DOI] [PubMed] [Google Scholar]
  10. Cáceres J. F., Krainer A. R. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 1993 Dec;12(12):4715–4726. doi: 10.1002/j.1460-2075.1993.tb06160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gozani O., Feld R., Reed R. Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. Genes Dev. 1996 Jan 15;10(2):233–243. doi: 10.1101/gad.10.2.233. [DOI] [PubMed] [Google Scholar]
  14. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  15. Henry M., Borland C. Z., Bossie M., Silver P. A. Potential RNA binding proteins in Saccharomyces cerevisiae identified as suppressors of temperature-sensitive mutations in NPL3. Genetics. 1996 Jan;142(1):103–115. doi: 10.1093/genetics/142.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higgins D. G., Thompson J. D., Gibson T. J. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 1996;266:383–402. doi: 10.1016/s0076-6879(96)66024-8. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  19. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  24. Nagai K., Oubridge C., Ito N., Avis J., Evans P. The RNP domain: a sequence-specific RNA-binding domain involved in processing and transport of RNA. Trends Biochem Sci. 1995 Jun;20(6):235–240. doi: 10.1016/s0968-0004(00)89024-6. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Peterson C. L., Dingwall A., Scott M. P. Five SWI/SNF gene products are components of a large multisubunit complex required for transcriptional enhancement. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2905–2908. doi: 10.1073/pnas.91.8.2905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ruiz-Lozano P., Doevendans P., Brown A., Gruber P. J., Chien K. R. Developmental expression of the murine spliceosome-associated protein mSAP49. Dev Dyn. 1997 Apr;208(4):482–490. doi: 10.1002/(SICI)1097-0177(199704)208:4<482::AID-AJA4>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  28. Sayle R. A., Milner-White E. J. RASMOL: biomolecular graphics for all. Trends Biochem Sci. 1995 Sep;20(9):374–374. doi: 10.1016/s0968-0004(00)89080-5. [DOI] [PubMed] [Google Scholar]
  29. Staknis D., Reed R. Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly. Mol Cell Biol. 1994 May;14(5):2994–3005. doi: 10.1128/mcb.14.5.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tanaka Y., Ohta A., Terashima K., Sakamoto H. Polycistronic expression and RNA-binding specificity of the C. elegans homologue of the spliceosome-associated protein SAP49. J Biochem. 1997 Apr;121(4):739–745. doi: 10.1093/oxfordjournals.jbchem.a021648. [DOI] [PubMed] [Google Scholar]
  31. VanHoy R. W., Wise J. A. Molecular analysis of a novel schizosaccharomyces pombe gene containing two RNP consensus-sequence RNA-binding domains. Curr Genet. 1996 Mar;29(4):307–315. doi: 10.1007/BF02208611. [DOI] [PubMed] [Google Scholar]
  32. Wells S. E., Neville M., Haynes M., Wang J., Igel H., Ares M., Jr CUS1, a suppressor of cold-sensitive U2 snRNA mutations, is a novel yeast splicing factor homologous to human SAP 145. Genes Dev. 1996 Jan 15;10(2):220–232. doi: 10.1101/gad.10.2.220. [DOI] [PubMed] [Google Scholar]
  33. Yan D., Ares M., Jr Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits. Mol Cell Biol. 1996 Mar;16(3):818–828. doi: 10.1128/mcb.16.3.818. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES