The role of U2AF35 and U2AF65 in enhancer-dependent splicing (original) (raw)

Abstract

Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.

Full Text

The Full Text of this article is available as a PDF (259.4 KB).

Selected References

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

  1. 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]
  2. Berglund J. A., Abovich N., Rosbash M. A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev. 1998 Mar 15;12(6):858–867. doi: 10.1101/gad.12.6.858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blencowe B. J. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci. 2000 Mar;25(3):106–110. doi: 10.1016/s0968-0004(00)01549-8. [DOI] [PubMed] [Google Scholar]
  4. Bouck J., Fu X. D., Skalka A. M., Katz R. A. Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants. J Biol Chem. 1998 Jun 12;273(24):15169–15176. doi: 10.1074/jbc.273.24.15169. [DOI] [PubMed] [Google Scholar]
  5. Burtis K. C., Baker B. S. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell. 1989 Mar 24;56(6):997–1010. doi: 10.1016/0092-8674(89)90633-8. [DOI] [PubMed] [Google Scholar]
  6. Eldridge A. G., Li Y., Sharp P. A., Blencowe B. J. The SRm160/300 splicing coactivator is required for exon-enhancer function. Proc Natl Acad Sci U S A. 1999 May 25;96(11):6125–6130. doi: 10.1073/pnas.96.11.6125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gama-Carvalho M., Krauss R. D., Chiang L., Valcárcel J., Green M. R., Carmo-Fonseca M. Targeting of U2AF65 to sites of active splicing in the nucleus. J Cell Biol. 1997 Jun 2;137(5):975–987. doi: 10.1083/jcb.137.5.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Graveley B. R., Hertel K. J., Maniatis T. A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers. EMBO J. 1998 Nov 16;17(22):6747–6756. doi: 10.1093/emboj/17.22.6747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Graveley B. R. Sorting out the complexity of SR protein functions. RNA. 2000 Sep;6(9):1197–1211. doi: 10.1017/s1355838200000960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guth S., Martínez C., Gaur R. K., Valcárcel J. Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65). Mol Cell Biol. 1999 Dec;19(12):8263–8271. doi: 10.1128/mcb.19.12.8263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoshijima K., Inoue K., Higuchi I., Sakamoto H., Shimura Y. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science. 1991 May 10;252(5007):833–836. doi: 10.1126/science.1902987. [DOI] [PubMed] [Google Scholar]
  14. Kan J. L., Green M. R. Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor. Genes Dev. 1999 Feb 15;13(4):462–471. doi: 10.1101/gad.13.4.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Konarska M. M. Analysis of splicing complexes and small nuclear ribonucleoprotein particles by native gel electrophoresis. Methods Enzymol. 1989;180:442–453. doi: 10.1016/0076-6879(89)80116-8. [DOI] [PubMed] [Google Scholar]
  17. Li Y., Blencowe B. J. Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract. J Biol Chem. 1999 Dec 3;274(49):35074–35079. doi: 10.1074/jbc.274.49.35074. [DOI] [PubMed] [Google Scholar]
  18. Lorson C. L., Androphy E. J. An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. Hum Mol Genet. 2000 Jan 22;9(2):259–265. doi: 10.1093/hmg/9.2.259. [DOI] [PubMed] [Google Scholar]
  19. Merendino L., Guth S., Bilbao D., Martínez C., Valcárcel J. Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG. Nature. 1999 Dec 16;402(6763):838–841. doi: 10.1038/45602. [DOI] [PubMed] [Google Scholar]
  20. Reed R. Mechanisms of fidelity in pre-mRNA splicing. Curr Opin Cell Biol. 2000 Jun;12(3):340–345. doi: 10.1016/s0955-0674(00)00097-1. [DOI] [PubMed] [Google Scholar]
  21. Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Rudner D. Z., Kanaar R., Breger K. S., Rio D. C. Interaction between subunits of heterodimeric splicing factor U2AF is essential in vivo. Mol Cell Biol. 1998 Apr;18(4):1765–1773. doi: 10.1128/mcb.18.4.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rudner D. Z., Kanaar R., Breger K. S., Rio D. C. Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10333–10337. doi: 10.1073/pnas.93.19.10333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith C. W., Valcárcel J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci. 2000 Aug;25(8):381–388. doi: 10.1016/s0968-0004(00)01604-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Tacke R., Manley J. L. Determinants of SR protein specificity. Curr Opin Cell Biol. 1999 Jun;11(3):358–362. doi: 10.1016/S0955-0674(99)80050-7. [DOI] [PubMed] [Google Scholar]
  29. Tian M., Maniatis T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 1994 Jul 15;8(14):1703–1712. doi: 10.1101/gad.8.14.1703. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Watakabe A., Tanaka K., Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993 Mar;7(3):407–418. doi: 10.1101/gad.7.3.407. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Wu S., Romfo C. M., Nilsen T. W., Green M. R. Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature. 1999 Dec 16;402(6763):832–835. doi: 10.1038/45590. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Zhang W. J., Wu J. Y. Functional properties of p54, a novel SR protein active in constitutive and alternative splicing. Mol Cell Biol. 1996 Oct;16(10):5400–5408. doi: 10.1128/mcb.16.10.5400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu J., Krainer A. R. Pre-mRNA splicing in the absence of an SR protein RS domain. Genes Dev. 2000 Dec 15;14(24):3166–3178. doi: 10.1101/gad.189500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zorio D. A., Blumenthal T. Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans. Nature. 1999 Dec 16;402(6763):835–838. doi: 10.1038/45597. [DOI] [PubMed] [Google Scholar]
  42. 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]