Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Jun 15;26(12):2955–2962. doi: 10.1093/nar/26.12.2955

Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor.

E Labourier 1, F Rossi 1, I E Gallouzi 1, E Allemand 1, G Divita 1, J Tazi 1
PMCID: PMC147637  PMID: 9611241

Abstract

Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase. Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats. These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation. Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF.

Full Text

The Full Text of this article is available as a PDF (340.3 KB).

Selected References

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

  1. Alsner J., Svejstrup J. Q., Kjeldsen E., Sørensen B. S., Westergaard O. Identification of an N-terminal domain of eukaryotic DNA topoisomerase I dispensable for catalytic activity but essential for in vivo function. J Biol Chem. 1992 Jun 25;267(18):12408–12411. [PubMed] [Google Scholar]
  2. 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]
  3. Bharti A. K., Olson M. O., Kufe D. W., Rubin E. H. Identification of a nucleolin binding site in human topoisomerase I. J Biol Chem. 1996 Jan 26;271(4):1993–1997. doi: 10.1074/jbc.271.4.1993. [DOI] [PubMed] [Google Scholar]
  4. Colwill K., Feng L. L., Yeakley J. M., Gish G. D., Cáceres J. F., Pawson T., Fu X. D. SRPK1 and Clk/Sty protein kinases show distinct substrate specificities for serine/arginine-rich splicing factors. J Biol Chem. 1996 Oct 4;271(40):24569–24575. doi: 10.1074/jbc.271.40.24569. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. D'Arpa P., Machlin P. S., Ratrie H., 3rd, Rothfield N. F., Cleveland D. W., Earnshaw W. C. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543–2547. doi: 10.1073/pnas.85.8.2543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Divita G., Goody R. S., Gautheron D. C., Di Pietro A. Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer. J Biol Chem. 1993 Jun 25;268(18):13178–13186. [PubMed] [Google Scholar]
  9. Fleischmann G., Pflugfelder G., Steiner E. K., Javaherian K., Howard G. C., Wang J. C., Elgin S. C. Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6958–6962. doi: 10.1073/pnas.81.22.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Gilmour D. S., Pflugfelder G., Wang J. C., Lis J. T. Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell. 1986 Feb 14;44(3):401–407. doi: 10.1016/0092-8674(86)90461-7. [DOI] [PubMed] [Google Scholar]
  13. Gobert C., Bracco L., Rossi F., Olivier M., Tazi J., Lavelle F., Larsen A. K., Riou J. F. Modulation of DNA topoisomerase I activity by p53. Biochemistry. 1996 May 7;35(18):5778–5786. doi: 10.1021/bi952327w. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gui J. F., Tronchère H., Chandler S. D., Fu X. D. Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10824–10828. doi: 10.1073/pnas.91.23.10824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gupta M., Fujimori A., Pommier Y. Eukaryotic DNA topoisomerases I. Biochim Biophys Acta. 1995 May 17;1262(1):1–14. doi: 10.1016/0167-4781(95)00029-g. [DOI] [PubMed] [Google Scholar]
  17. Heck M. M., Hittelman W. N., Earnshaw W. C. Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1086–1090. doi: 10.1073/pnas.85.4.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jiménez-García L. F., Spector D. L. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell. 1993 Apr 9;73(1):47–59. doi: 10.1016/0092-8674(93)90159-n. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Kretzschmar M., Meisterernst M., Roeder R. G. Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11508–11512. doi: 10.1073/pnas.90.24.11508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Liu L. F., Miller K. G. Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3487–3491. doi: 10.1073/pnas.78.6.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Merino A., Madden K. R., Lane W. S., Champoux J. J., Reinberg D. DNA topoisomerase I is involved in both repression and activation of transcription. Nature. 1993 Sep 16;365(6443):227–232. doi: 10.1038/365227a0. [DOI] [PubMed] [Google Scholar]
  26. Parker A. R., Steitz J. A. Inhibition of mammalian spliceosome assembly and pre-mRNA splicing by peptide inhibitors of protein kinases. RNA. 1997 Nov;3(11):1301–1312. [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Rittinger K., Divita G., Goody R. S. Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):8046–8049. doi: 10.1073/pnas.92.17.8046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rose K. M., Szopa J., Han F. S., Cheng Y. C., Richter A., Scheer U. Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription. Chromosoma. 1988;96(6):411–416. doi: 10.1007/BF00303034. [DOI] [PubMed] [Google Scholar]
  31. Rossi F., Labourier E., Forné T., Divita G., Derancourt J., Riou J. F., Antoine E., Cathala G., Brunel C., Tazi J. Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature. 1996 May 2;381(6577):80–82. doi: 10.1038/381080a0. [DOI] [PubMed] [Google Scholar]
  32. Roth M. B., Gall J. G. Monoclonal antibodies that recognize transcription unit proteins on newt lampbrush chromosomes. J Cell Biol. 1987 Sep;105(3):1047–1054. doi: 10.1083/jcb.105.3.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Shykind B. M., Kim J., Stewart L., Champoux J. J., Sharp P. A. Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription. Genes Dev. 1997 Feb 1;11(3):397–407. doi: 10.1101/gad.11.3.397. [DOI] [PubMed] [Google Scholar]
  36. Spector D. L. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. doi: 10.1146/annurev.cb.09.110193.001405. [DOI] [PubMed] [Google Scholar]
  37. Stewart L., Ireton G. C., Champoux J. J. The domain organization of human topoisomerase I. J Biol Chem. 1996 Mar 29;271(13):7602–7608. doi: 10.1074/jbc.271.13.7602. [DOI] [PubMed] [Google Scholar]
  38. Stewart L., Ireton G. C., Champoux J. J. The domain organization of human topoisomerase I. J Biol Chem. 1996 Mar 29;271(13):7602–7608. doi: 10.1074/jbc.271.13.7602. [DOI] [PubMed] [Google Scholar]
  39. Stewart L., Ireton G. C., Parker L. H., Madden K. R., Champoux J. J. Biochemical and biophysical analyses of recombinant forms of human topoisomerase I. J Biol Chem. 1996 Mar 29;271(13):7593–7601. doi: 10.1074/jbc.271.13.7593. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Theissen H., Etzerodt M., Reuter R., Schneider C., Lottspeich F., Argos P., Lührmann R., Philipson L. Cloning of the human cDNA for the U1 RNA-associated 70K protein. EMBO J. 1986 Dec 1;5(12):3209–3217. doi: 10.1002/j.1460-2075.1986.tb04631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Woppmann A., Patschinsky T., Bringmann P., Godt F., Lührmann R. Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants. Nucleic Acids Res. 1990 Aug 11;18(15):4427–4438. doi: 10.1093/nar/18.15.4427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Woppmann A., Will C. L., Kornstädt U., Zuo P., Manley J. L., Lührmann R. Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors. Nucleic Acids Res. 1993 Jun 25;21(12):2815–2822. doi: 10.1093/nar/21.12.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Wuarin J., Schibler U. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol Cell Biol. 1994 Nov;14(11):7219–7225. doi: 10.1128/mcb.14.11.7219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]
  53. Zuo P., Manley J. L. Functional domains of the human splicing factor ASF/SF2. EMBO J. 1993 Dec;12(12):4727–4737. doi: 10.1002/j.1460-2075.1993.tb06161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES