Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Aug;17(8):4667–4676. doi: 10.1128/mcb.17.8.4667

The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

R C Chan 1, D L Black 1
PMCID: PMC232319  PMID: 9234723

Abstract

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

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

  1. Adams M. D., Rudner D. Z., Rio D. C. Biochemistry and regulation of pre-mRNA splicing. Curr Opin Cell Biol. 1996 Jun;8(3):331–339. doi: 10.1016/s0955-0674(96)80006-8. [DOI] [PubMed] [Google Scholar]
  2. Amendt B. A., Si Z. H., Stoltzfus C. M. Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors. Mol Cell Biol. 1995 Aug;15(8):4606–4615. doi: 10.1128/mcb.15.8.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balvay L., Libri D., Gallego M., Fiszman M. Y. Intronic sequence with both negative and positive effects on the regulation of alternative transcripts of the chicken beta tropomyosin transcripts. Nucleic Acids Res. 1992 Aug 11;20(15):3987–3992. doi: 10.1093/nar/20.15.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bell L. R., Horabin J. I., Schedl P., Cline T. W. Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell. 1991 Apr 19;65(2):229–239. doi: 10.1016/0092-8674(91)90157-t. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Black D. L. Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro. Cell. 1992 May 29;69(5):795–807. doi: 10.1016/0092-8674(92)90291-j. [DOI] [PubMed] [Google Scholar]
  7. Black D. L. Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells? Genes Dev. 1991 Mar;5(3):389–402. doi: 10.1101/gad.5.3.389. [DOI] [PubMed] [Google Scholar]
  8. Black D. L. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PMC free article] [PubMed] [Google Scholar]
  9. Boggs R. T., Gregor P., Idriss S., Belote J. M., McKeown M. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell. 1987 Aug 28;50(5):739–747. doi: 10.1016/0092-8674(87)90332-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Butler B., Pirrotta V., Irminger-Finger I., Nöthiger R. The sex-determining gene tra of Drosophila: molecular cloning and transformation studies. EMBO J. 1986 Dec 20;5(13):3607–3613. doi: 10.1002/j.1460-2075.1986.tb04689.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Caputi M., Casari G., Guenzi S., Tagliabue R., Sidoli A., Melo C. A., Baralle F. E. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon. Nucleic Acids Res. 1994 Mar 25;22(6):1018–1022. doi: 10.1093/nar/22.6.1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chabot B. Directing alternative splicing: cast and scenarios. Trends Genet. 1996 Nov;12(11):472–478. doi: 10.1016/0168-9525(96)10037-8. [DOI] [PubMed] [Google Scholar]
  14. Chan R. C., Black D. L. Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro. Mol Cell Biol. 1995 Nov;15(11):6377–6385. doi: 10.1128/mcb.15.11.6377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Del Gatto F., Breathnach R. Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon. Mol Cell Biol. 1995 Sep;15(9):4825–4834. doi: 10.1128/mcb.15.9.4825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deshpande G., Samuels M. E., Schedl P. D. Sex-lethal interacts with splicing factors in vitro and in vivo. Mol Cell Biol. 1996 Sep;16(9):5036–5047. doi: 10.1128/mcb.16.9.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dominski Z., Kole R. Selection of splice sites in pre-mRNAs with short internal exons. Mol Cell Biol. 1991 Dec;11(12):6075–6083. doi: 10.1128/mcb.11.12.6075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Estes P. A., Cooke N. E., Liebhaber S. A. A native RNA secondary structure controls alternative splice-site selection and generates two human growth hormone isoforms. J Biol Chem. 1992 Jul 25;267(21):14902–14908. [PubMed] [Google Scholar]
  19. García-Blanco M. A., Jamison S. F., Sharp P. A. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 1989 Dec;3(12A):1874–1886. doi: 10.1101/gad.3.12a.1874. [DOI] [PubMed] [Google Scholar]
  20. Ghetti A., Piñol-Roma S., Michael W. M., Morandi C., Dreyfuss G. hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res. 1992 Jul 25;20(14):3671–3678. doi: 10.1093/nar/20.14.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gooding C., Roberts G. C., Moreau G., Nadal-Ginard B., Smith C. W. Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon. EMBO J. 1994 Aug 15;13(16):3861–3872. doi: 10.1002/j.1460-2075.1994.tb06697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Graham I. R., Hamshere M., Eperon I. C. Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences. Mol Cell Biol. 1992 Sep;12(9):3872–3882. doi: 10.1128/mcb.12.9.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Guo W., Mulligan G. J., Wormsley S., Helfman D. M. Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells. Genes Dev. 1991 Nov;5(11):2096–2107. doi: 10.1101/gad.5.11.2096. [DOI] [PubMed] [Google Scholar]
  24. Helfman D. M., Ricci W. M., Finn L. A. Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo. Genes Dev. 1988 Dec;2(12A):1627–1638. doi: 10.1101/gad.2.12a.1627. [DOI] [PubMed] [Google Scholar]
  25. Horabin J. I., Schedl P. Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5' splice site. Mol Cell Biol. 1993 Dec;13(12):7734–7746. doi: 10.1128/mcb.13.12.7734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Inoue K., Hoshijima K., Sakamoto H., Shimura Y. Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript. Nature. 1990 Mar 29;344(6265):461–463. doi: 10.1038/344461a0. [DOI] [PubMed] [Google Scholar]
  27. Kanopka A., Mühlemann O., Akusjärvi G. Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA. Nature. 1996 Jun 6;381(6582):535–538. doi: 10.1038/381535a0. [DOI] [PubMed] [Google Scholar]
  28. Levy J. B., Dorai T., Wang L. H., Brugge J. S. The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA. Mol Cell Biol. 1987 Nov;7(11):4142–4145. doi: 10.1128/mcb.7.11.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Libri D., Balvay L., Fiszman M. Y. In vivo splicing of the beta tropomyosin pre-mRNA: a role for branch point and donor site competition. Mol Cell Biol. 1992 Jul;12(7):3204–3215. doi: 10.1128/mcb.12.7.3204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Libri D., Piseri A., Fiszman M. Y. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure. Science. 1991 Jun 28;252(5014):1842–1845. doi: 10.1126/science.2063196. [DOI] [PubMed] [Google Scholar]
  31. Lin C. H., Patton J. G. Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA. 1995 May;1(3):234–245. [PMC free article] [PubMed] [Google Scholar]
  32. Lou H., Gagel R. F., Berget S. M. An intron enhancer recognized by splicing factors activates polyadenylation. Genes Dev. 1996 Jan 15;10(2):208–219. doi: 10.1101/gad.10.2.208. [DOI] [PubMed] [Google Scholar]
  33. Martinez R., Mathey-Prevot B., Bernards A., Baltimore D. Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. Science. 1987 Jul 24;237(4813):411–415. doi: 10.1126/science.2440106. [DOI] [PubMed] [Google Scholar]
  34. Mayeda A., Munroe S. H., Cáceres J. F., Krainer A. R. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. 1994 Nov 15;13(22):5483–5495. doi: 10.1002/j.1460-2075.1994.tb06883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McKeown M. Sex differentiation: the role of alternative splicing. Curr Opin Genet Dev. 1992 Apr;2(2):299–303. doi: 10.1016/s0959-437x(05)80288-6. [DOI] [PubMed] [Google Scholar]
  36. Min H., Chan R. C., Black D. L. The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event. Genes Dev. 1995 Nov 1;9(21):2659–2671. doi: 10.1101/gad.9.21.2659. [DOI] [PubMed] [Google Scholar]
  37. Mulligan G. J., Guo W., Wormsley S., Helfman D. M. Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA. J Biol Chem. 1992 Dec 15;267(35):25480–25487. [PubMed] [Google Scholar]
  38. Nemeroff M. E., Utans U., Krämer A., Krug R. M. Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes. Mol Cell Biol. 1992 Mar;12(3):962–970. doi: 10.1128/mcb.12.3.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Norton P. A., Hynes R. O. Characterization of HeLa nuclear factors which interact with a conditionally processed rat fibronectin pre-mRNA. Biochem Biophys Res Commun. 1993 Aug 31;195(1):215–221. doi: 10.1006/bbrc.1993.2032. [DOI] [PubMed] [Google Scholar]
  40. O'Reilly M. M., McNally M. T., Beemon K. L. Two strong 5' splice sites and competing, suboptimal 3' splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA. Virology. 1995 Nov 10;213(2):373–385. doi: 10.1006/viro.1995.0010. [DOI] [PubMed] [Google Scholar]
  41. Patton J. G., Mayer S. A., Tempst P., Nadal-Ginard B. Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing. Genes Dev. 1991 Jul;5(7):1237–1251. doi: 10.1101/gad.5.7.1237. [DOI] [PubMed] [Google Scholar]
  42. Patton J. G., Porro E. B., Galceran J., Tempst P., Nadal-Ginard B. Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Genes Dev. 1993 Mar;7(3):393–406. doi: 10.1101/gad.7.3.393. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Sakamoto H., Inoue K., Higuchi I., Ono Y., Shimura Y. Control of Drosophila Sex-lethal pre-mRNA splicing by its own female-specific product. Nucleic Acids Res. 1992 Nov 11;20(21):5533–5540. doi: 10.1093/nar/20.21.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Samuels M. E., Bopp D., Colvin R. A., Roscigno R. F., Garcia-Blanco M. A., Schedl P. RNA binding by Sxl proteins in vitro and in vivo. Mol Cell Biol. 1994 Jul;14(7):4975–4990. doi: 10.1128/mcb.14.7.4975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Siebel C. W., Fresco L. D., Rio D. C. The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding. Genes Dev. 1992 Aug;6(8):1386–1401. doi: 10.1101/gad.6.8.1386. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Sirand-Pugnet P., Durosay P., Clouet d'Orval B. C., Brody E., Marie J. beta-Tropomyosin pre-mRNA folding around a muscle-specific exon interferes with several steps of spliceosome assembly. J Mol Biol. 1995 Sep 1;251(5):591–602. doi: 10.1006/jmbi.1995.0458. [DOI] [PubMed] [Google Scholar]
  49. Sosnowski B. A., Belote J. M., McKeown M. Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell. 1989 Aug 11;58(3):449–459. doi: 10.1016/0092-8674(89)90426-1. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Swanson M. S., Dreyfuss G. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol. 1988 May;8(5):2237–2241. doi: 10.1128/mcb.8.5.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Swanson M. S., Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES