iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170960
Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2 - PMC Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Nov 2;17(21):6359–6367. doi: 10.1093/emboj/17.21.6359

Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2.

S H Xiao 1, J L Manley 1
PMCID: PMC1170960  PMID: 9799243

Abstract

SR proteins are a conserved family of splicing factors that function in both constitutive and activated splicing. We reported previously that phosphorylation of the SR protein ASF/SF2 enhances its interaction with the U1 snRNP-specific 70K protein and is required for the protein to function in splicing, while other studies have provided evidence that subsequent dephosphorylation can also be required for SR protein function, at least in constitutive splicing. We now show that the phosphorylation status of ASF/SF2 can differentially affect several properties of the protein. In keeping with a dynamic cycle of phosphorylation-dephosphorylation during splicing, ASF/SF2 phosphorylation was found to affect interaction with several putative protein targets in different ways: positively, negatively or not at all. Extending these results, we also show that, in contrast to constitutive splicing, dephosphorylation is not required for ASF/SF2 to function as a splicing activator. We discuss these results with respect to the differential protein-protein interactions that must occur during constitutive and activated splicing.

Full Text

The Full Text of this article is available as a PDF (222.4 KB).

Selected References

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

  1. Amrein H., Hedley M. L., Maniatis T. The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2. Cell. 1994 Feb 25;76(4):735–746. doi: 10.1016/0092-8674(94)90512-6. [DOI] [PubMed] [Google Scholar]
  2. Berget S. M. Exon recognition in vertebrate splicing. J Biol Chem. 1995 Feb 10;270(6):2411–2414. doi: 10.1074/jbc.270.6.2411. [DOI] [PubMed] [Google Scholar]
  3. Cao W., Jamison S. F., Garcia-Blanco M. A. Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro. RNA. 1997 Dec;3(12):1456–1467. [PMC free article] [PubMed] [Google Scholar]
  4. Champion-Arnaud P., Gozani O., Palandjian L., Reed R. Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations. Mol Cell Biol. 1995 Oct;15(10):5750–5756. doi: 10.1128/mcb.15.10.5750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chandler S. D., Mayeda A., Yeakley J. M., Krainer A. R., Fu X. D. RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3596–3601. doi: 10.1073/pnas.94.8.3596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colwill K., Pawson T., Andrews B., Prasad J., Manley J. L., Bell J. C., Duncan P. I. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 1996 Jan 15;15(2):265–275. [PMC free article] [PubMed] [Google Scholar]
  7. Crispino J. D., Blencowe B. J., Sharp P. A. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science. 1994 Sep 23;265(5180):1866–1869. doi: 10.1126/science.8091213. [DOI] [PubMed] [Google Scholar]
  8. Crispino J. D., Sharp P. A. A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. Genes Dev. 1995 Sep 15;9(18):2314–2323. doi: 10.1101/gad.9.18.2314. [DOI] [PubMed] [Google Scholar]
  9. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  10. Eperon I. C., Ireland D. C., Smith R. A., Mayeda A., Krainer A. R. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993 Sep;12(9):3607–3617. doi: 10.1002/j.1460-2075.1993.tb06034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fu X. D., Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science. 1992 Apr 24;256(5056):535–538. doi: 10.1126/science.1373910. [DOI] [PubMed] [Google Scholar]
  12. Fu X. D., Mayeda A., Maniatis T., Krainer A. R. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11224–11228. doi: 10.1073/pnas.89.23.11224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fu X. D. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. doi: 10.1038/365082a0. [DOI] [PubMed] [Google Scholar]
  14. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  15. Ge H., Manley J. L. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell. 1990 Jul 13;62(1):25–34. doi: 10.1016/0092-8674(90)90236-8. [DOI] [PubMed] [Google Scholar]
  16. Ge H., Zuo P., Manley J. L. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. doi: 10.1016/0092-8674(91)90626-a. [DOI] [PubMed] [Google Scholar]
  17. Graveley B. R., Maniatis T. Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing. Mol Cell. 1998 Apr;1(5):765–771. doi: 10.1016/s1097-2765(00)80076-3. [DOI] [PubMed] [Google Scholar]
  18. Gui J. F., Lane W. S., Fu X. D. A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature. 1994 Jun 23;369(6482):678–682. doi: 10.1038/369678a0. [DOI] [PubMed] [Google Scholar]
  19. Huang S., Spector D. L. Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription. J Cell Biol. 1996 May;133(4):719–732. doi: 10.1083/jcb.133.4.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jamison S. F., Pasman Z., Wang J., Will C., Lührmann R., Manley J. L., Garcia-Blanco M. A. U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements. Nucleic Acids Res. 1995 Aug 25;23(16):3260–3267. doi: 10.1093/nar/23.16.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  22. Konforti B. B., Koziolkiewicz M. J., Konarska M. M. Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly. Cell. 1993 Dec 3;75(5):863–873. doi: 10.1016/0092-8674(93)90531-t. [DOI] [PubMed] [Google Scholar]
  23. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  24. Krainer A. R., Conway G. C., Kozak D. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites. Cell. 1990 Jul 13;62(1):35–42. doi: 10.1016/0092-8674(90)90237-9. [DOI] [PubMed] [Google Scholar]
  25. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lynch K. W., Maniatis T. Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes Dev. 1996 Aug 15;10(16):2089–2101. doi: 10.1101/gad.10.16.2089. [DOI] [PubMed] [Google Scholar]
  28. Lynch K. W., Maniatis T. Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 1995 Feb 1;9(3):284–293. doi: 10.1101/gad.9.3.284. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Mermoud J. E., Cohen P. T., Lamond A. I. Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J. 1994 Dec 1;13(23):5679–5688. doi: 10.1002/j.1460-2075.1994.tb06906.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mermoud J. E., Cohen P., Lamond A. I. Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res. 1992 Oct 25;20(20):5263–5269. doi: 10.1093/nar/20.20.5263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Misteli T., Cáceres J. F., Spector D. L. The dynamics of a pre-mRNA splicing factor in living cells. Nature. 1997 May 29;387(6632):523–527. doi: 10.1038/387523a0. [DOI] [PubMed] [Google Scholar]
  34. Misteli T., Spector D. L. Protein phosphorylation and the nuclear organization of pre-mRNA splicing. Trends Cell Biol. 1997 Apr;7(4):135–138. doi: 10.1016/S0962-8924(96)20043-1. [DOI] [PubMed] [Google Scholar]
  35. Ramchatesingh J., Zahler A. M., Neugebauer K. M., Roth M. B., Cooper T. A. A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer. Mol Cell Biol. 1995 Sep;15(9):4898–4907. doi: 10.1128/mcb.15.9.4898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Roth M. B., Murphy C., Gall J. G. A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle. J Cell Biol. 1990 Dec;111(6 Pt 1):2217–2223. doi: 10.1083/jcb.111.6.2217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Si Z., Amendt B. A., Stoltzfus C. M. Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2. Nucleic Acids Res. 1997 Feb 15;25(4):861–867. doi: 10.1093/nar/25.4.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Singer R. H., Green M. R. Compartmentalization of eukaryotic gene expression: causes and effects. Cell. 1997 Oct 31;91(3):291–294. doi: 10.1016/s0092-8674(00)80411-0. [DOI] [PubMed] [Google Scholar]
  41. Staffa A., Cochrane A. Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1. Mol Cell Biol. 1995 Aug;15(8):4597–4605. doi: 10.1128/mcb.15.8.4597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Staffa A., Cochrane A. The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site. J Virol. 1994 May;68(5):3071–3079. doi: 10.1128/jvi.68.5.3071-3079.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Staknis D., Reed R. Members of a family of proteins (the RD family) detected by a U1 70K monoclonal antibody are present in spliceosomal complexes. Nucleic Acids Res. 1995 Oct 25;23(20):4081–4086. doi: 10.1093/nar/23.20.4081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Staknis D., Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. doi: 10.1128/mcb.14.11.7670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sun Q., Mayeda A., Hampson R. K., Krainer A. R., Rottman F. M. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. doi: 10.1101/gad.7.12b.2598. [DOI] [PubMed] [Google Scholar]
  46. Tacke R., Chen Y., Manley J. L. Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1148–1153. doi: 10.1073/pnas.94.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tacke R., Manley J. L. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995 Jul 17;14(14):3540–3551. doi: 10.1002/j.1460-2075.1995.tb07360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tacke R., Tohyama M., Ogawa S., Manley J. L. Human Tra2 proteins are sequence-specific activators of pre-mRNA splicing. Cell. 1998 Apr 3;93(1):139–148. doi: 10.1016/s0092-8674(00)81153-8. [DOI] [PubMed] [Google Scholar]
  49. Tarn W. Y., Steitz J. A. Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2504–2508. doi: 10.1073/pnas.92.7.2504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tazi J., Daugeron M. C., Cathala G., Brunel C., Jeanteur P. Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing. J Biol Chem. 1992 Mar 5;267(7):4322–4326. [PubMed] [Google Scholar]
  51. Tazi J., Kornstädt U., Rossi F., Jeanteur P., Cathala G., Brunel C., Lührmann R. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 1993 May 20;363(6426):283–286. doi: 10.1038/363283a0. [DOI] [PubMed] [Google Scholar]
  52. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Wang J., Manley J. L. Overexpression of the SR proteins ASF/SF2 and SC35 influences alternative splicing in vivo in diverse ways. RNA. 1995 May;1(3):335–346. [PMC free article] [PubMed] [Google Scholar]
  56. Wang J., Xiao S. H., Manley J. L. Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing. Genes Dev. 1998 Jul 15;12(14):2222–2233. doi: 10.1101/gad.12.14.2222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wang Z., Hoffmann H. M., Grabowski P. J. Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity. RNA. 1995 Mar;1(1):21–35. [PMC free article] [PubMed] [Google Scholar]
  58. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  59. Xiao S. H., Manley J. L. Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes Dev. 1997 Feb 1;11(3):334–344. doi: 10.1101/gad.11.3.334. [DOI] [PubMed] [Google Scholar]
  60. Yeakley J. M., Morfin J. P., Rosenfeld M. G., Fu X. D. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7582–7587. doi: 10.1073/pnas.93.15.7582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]
  62. Zahler A. M., Neugebauer K. M., Lane W. S., Roth M. B. Distinct functions of SR proteins in alternative pre-mRNA splicing. Science. 1993 Apr 9;260(5105):219–222. doi: 10.1126/science.8385799. [DOI] [PubMed] [Google Scholar]
  63. Zahler A. M., Roth M. B. Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2642–2646. doi: 10.1073/pnas.92.7.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. 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]
  65. Zeng C., Kim E., Warren S. L., Berget S. M. Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity. EMBO J. 1997 Mar 17;16(6):1401–1412. doi: 10.1093/emboj/16.6.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhang G., Taneja K. L., Singer R. H., Green M. R. Localization of pre-mRNA splicing in mammalian nuclei. Nature. 1994 Dec 22;372(6508):809–812. doi: 10.1038/372809a0. [DOI] [PubMed] [Google Scholar]
  67. 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]
  68. 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]
  69. Zuo P., Manley J. L. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. doi: 10.1073/pnas.91.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES