Skip to main content
PMC home page
RNA logoLink to RNA
. 1999 May;5(5):687–706. doi: 10.1017/s1355838299990155

Combinatorial control of a neuron-specific exon.

E F Modafferi 1, D L Black 1
PMCID: PMC1369796  PMID: 10334339

Abstract

The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3' splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3' splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3' splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Ashiya M., Grabowski P. J. A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart. RNA. 1997 Sep;3(9):996–1015. [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Brown M. T., Cooper J. A. Regulation, substrates and functions of src. Biochim Biophys Acta. 1996 Jun 7;1287(2-3):121–149. doi: 10.1016/0304-419x(96)00003-0. [DOI] [PubMed] [Google Scholar]
  6. Buckanovich R. J., Darnell R. B. The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo. Mol Cell Biol. 1997 Jun;17(6):3194–3201. doi: 10.1128/mcb.17.6.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlo T., Sterner D. A., Berget S. M. An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon. RNA. 1996 Apr;2(4):342–353. [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Chan R. C., Black D. L. The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream. Mol Cell Biol. 1997 Aug;17(8):4667–4676. doi: 10.1128/mcb.17.8.4667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chou M. Y., Rooke N., Turck C. W., Black D. L. hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells. Mol Cell Biol. 1999 Jan;19(1):69–77. doi: 10.1128/mcb.19.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cramer P., Pesce C. G., Baralle F. E., Kornblihtt A. R. Functional association between promoter structure and transcript alternative splicing. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11456–11460. doi: 10.1073/pnas.94.21.11456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Dominski Z., Kole R. Cooperation of pre-mRNA sequence elements in splice site selection. Mol Cell Biol. 1992 May;12(5):2108–2114. doi: 10.1128/mcb.12.5.2108. [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. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  19. Ernst P., Smale S. T. Combinatorial regulation of transcription II: The immunoglobulin mu heavy chain gene. Immunity. 1995 May;2(5):427–438. doi: 10.1016/1074-7613(95)90024-1. [DOI] [PubMed] [Google Scholar]
  20. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [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. Gooding C., Roberts G. C., Smith C. W. Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon. RNA. 1998 Jan;4(1):85–100. [PMC free article] [PubMed] [Google Scholar]
  23. Helfman D. M. The generation of protein isoform diversity by alternative RNA splicing. Soc Gen Physiol Ser. 1994;49:105–115. [PubMed] [Google Scholar]
  24. Hertel K. J., Lynch K. W., Maniatis T. Common themes in the function of transcription and splicing enhancers. Curr Opin Cell Biol. 1997 Jun;9(3):350–357. doi: 10.1016/s0955-0674(97)80007-5. [DOI] [PubMed] [Google Scholar]
  25. Hertel K. J., Maniatis T. The function of multisite splicing enhancers. Mol Cell. 1998 Feb;1(3):449–455. doi: 10.1016/s1097-2765(00)80045-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Huh G. S., Hynes R. O. Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. Genes Dev. 1994 Jul 1;8(13):1561–1574. doi: 10.1101/gad.8.13.1561. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Kamma H., Portman D. S., Dreyfuss G. Cell type-specific expression of hnRNP proteins. Exp Cell Res. 1995 Nov;221(1):187–196. doi: 10.1006/excr.1995.1366. [DOI] [PubMed] [Google Scholar]
  30. Kawamoto S. Neuron-specific alternative splicing of nonmuscle myosin II heavy chain-B pre-mRNA requires a cis-acting intron sequence. J Biol Chem. 1996 Jul 26;271(30):17613–17616. [PubMed] [Google Scholar]
  31. Koushika S. P., Lisbin M. J., White K. ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform. Curr Biol. 1996 Dec 1;6(12):1634–1641. doi: 10.1016/s0960-9822(02)70787-2. [DOI] [PubMed] [Google Scholar]
  32. Lavigueur A., La Branche H., Kornblihtt A. R., Chabot B. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 1993 Dec;7(12A):2405–2417. doi: 10.1101/gad.7.12a.2405. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Lim L. P., Sharp P. A. Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats. Mol Cell Biol. 1998 Jul;18(7):3900–3906. doi: 10.1128/mcb.18.7.3900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Liu H. X., Zhang M., Krainer A. R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 1998 Jul 1;12(13):1998–2012. doi: 10.1101/gad.12.13.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Mayeda A., Zahler A. M., Krainer A. R., Roth M. B. Two members of a conserved family of nuclear phosphoproteins are involved in pre-mRNA splicing. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1301–1304. doi: 10.1073/pnas.89.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Min H., Turck C. W., Nikolic J. M., Black D. L. A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes Dev. 1997 Apr 15;11(8):1023–1036. doi: 10.1101/gad.11.8.1023. [DOI] [PubMed] [Google Scholar]
  42. Modafferi E. F., Black D. L. A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon. Mol Cell Biol. 1997 Nov;17(11):6537–6545. doi: 10.1128/mcb.17.11.6537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Norton P. A. Polypyrimidine tract sequences direct selection of alternative branch sites and influence protein binding. Nucleic Acids Res. 1994 Sep 25;22(19):3854–3860. doi: 10.1093/nar/22.19.3854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Ryan K. J., Cooper T. A. Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle. Mol Cell Biol. 1996 Aug;16(8):4014–4023. doi: 10.1128/mcb.16.8.4014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Santoro B., Grant S. G., Bartsch D., Kandel E. R. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14815–14820. doi: 10.1073/pnas.94.26.14815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schaal T. D., Maniatis T. Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA. Mol Cell Biol. 1999 Jan;19(1):261–273. doi: 10.1128/mcb.19.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Sirand-Pugnet P., Durosay P., Brody E., Marie J. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA. Nucleic Acids Res. 1995 Sep 11;23(17):3501–3507. doi: 10.1093/nar/23.17.3501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. 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]
  57. 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]
  58. 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]
  59. Valcárcel J., Gebauer F. Post-transcriptional regulation: the dawn of PTB. Curr Biol. 1997 Nov 1;7(11):R705–R708. doi: 10.1016/s0960-9822(06)00361-7. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. 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]
  62. Wang J., Dong Z., Bell L. R. Sex-lethal interactions with protein and RNA. Roles of glycine-rich and RNA binding domains. J Biol Chem. 1997 Aug 29;272(35):22227–22235. doi: 10.1074/jbc.272.35.22227. [DOI] [PubMed] [Google Scholar]
  63. Wang J., Manley J. L. Regulation of pre-mRNA splicing in metazoa. Curr Opin Genet Dev. 1997 Apr;7(2):205–211. doi: 10.1016/s0959-437x(97)80130-x. [DOI] [PubMed] [Google Scholar]
  64. 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]
  65. Wei N., Lin C. Q., Modafferi E. F., Gomes W. A., Black D. L. A unique intronic splicing enhancer controls the inclusion of the agrin Y exon. RNA. 1997 Nov;3(11):1275–1288. [PMC free article] [PubMed] [Google Scholar]
  66. 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]
  67. 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]
  68. Zhang L., Ashiya M., Sherman T. G., Grabowski P. J. Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA. RNA. 1996 Jul;2(7):682–698. [PMC free article] [PubMed] [Google Scholar]
  69. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES