cis-regulatory sequences responsible for alternative splicing of the Drosophila dopa decarboxylase gene (original) (raw)

Abstract

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in specific sets of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. The Ddc CNS mRNA contains all four exons (A through D), whereas the hypodermal mRNA contains only three exons (A, C, and D). To localize cis-regulatory sequences responsible for Ddc alternative splicing, a Ddc minigene and several fusion genes containing various amounts of Ddc sequences fused to fushi tarazu (ftz) exon 1 were constructed and introduced into flies by P-element-mediated germ line transformation. We find that Ddc intron ab and exon B are sufficient to regulate Ddc alternative splicing, since transcripts of a minimal fusion gene containing most of Ddc intron ab and exon B are spliced to exon B in the CNS but not in the hypoderm. These results indicate that Ddc alternative splicing is regulated by either a negative mechanism preventing splicing to exon B in the hypoderm or a positive mechanism activating splicing to exon B in the CNS. Our previous data suggest that Ddc hypodermal splicing is the actively regulated splicing pathway (J. Shen, C. J. Beall, and J. Hirsh, Mol. Cell. Biol. 13:4549-4555, 1993). Here we show that deletion of Ddc intron ab sequences selectively disrupts hypodermal splicing specificity. These results support a model in which Ddc alternative splicing is negatively regulated by a blockage mechanism preventing splicing to exon B in the hypoderm.

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Selected References

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  1. Beall C. J., Hirsh J. Regulation of the Drosophila dopa decarboxylase gene in neuronal and glial cells. Genes Dev. 1987 Jul;1(5):510–520. doi: 10.1101/gad.1.5.510. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bell L. R., Maine E. M., Schedl P., Cline T. W. Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell. 1988 Dec 23;55(6):1037–1046. doi: 10.1016/0092-8674(88)90248-6. [DOI] [PubMed] [Google Scholar]
  4. Bingham P. M., Chou T. B., Mims I., Zachar Z. On/off regulation of gene expression at the level of splicing. Trends Genet. 1988 May;4(5):134–138. doi: 10.1016/0168-9525(88)90136-9. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Bray S. J., Johnson W. A., Hirsh J., Heberlein U., Tjian R. A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene. EMBO J. 1988 Jan;7(1):177–188. doi: 10.1002/j.1460-2075.1988.tb02798.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  11. Emeson R. B., Hedjran F., Yeakley J. M., Guise J. W., Rosenfeld M. G. Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor. Nature. 1989 Sep 7;341(6237):76–80. doi: 10.1038/341076a0. [DOI] [PubMed] [Google Scholar]
  12. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  13. Hedley M. L., Maniatis T. Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro. Cell. 1991 May 17;65(4):579–586. doi: 10.1016/0092-8674(91)90090-l. [DOI] [PubMed] [Google Scholar]
  14. Horabin J. I., Schedl P. Regulated splicing of the Drosophila sex-lethal male exon involves a blockage mechanism. Mol Cell Biol. 1993 Mar;13(3):1408–1414. doi: 10.1128/mcb.13.3.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hortsch M., Bieber A. J., Patel N. H., Goodman C. S. Differential splicing generates a nervous system-specific form of Drosophila neuroglian. Neuron. 1990 May;4(5):697–709. doi: 10.1016/0896-6273(90)90196-m. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Karess R. E., Rubin G. M. Analysis of P transposable element functions in Drosophila. Cell. 1984 Aug;38(1):135–146. doi: 10.1016/0092-8674(84)90534-8. [DOI] [PubMed] [Google Scholar]
  19. Konrad K. D., Marsh J. L. Developmental expression and spatial distribution of dopa decarboxylase in Drosophila. Dev Biol. 1987 Jul;122(1):172–185. doi: 10.1016/0012-1606(87)90343-5. [DOI] [PubMed] [Google Scholar]
  20. Kornfeld K., Saint R. B., Beachy P. A., Harte P. J., Peattie D. A., Hogness D. S. Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila. Genes Dev. 1989 Feb;3(2):243–258. doi: 10.1101/gad.3.2.243. [DOI] [PubMed] [Google Scholar]
  21. Laski F. A., Rubin G. M. Analysis of the cis-acting requirements for germ-line-specific splicing of the P-element ORF2-ORF3 intron. Genes Dev. 1989 May;3(5):720–728. doi: 10.1101/gad.3.5.720. [DOI] [PubMed] [Google Scholar]
  22. Maniatis T. Mechanisms of alternative pre-mRNA splicing. Science. 1991 Jan 4;251(4989):33–34. doi: 10.1126/science.1824726. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. McKeown M. Alternative mRNA splicing. Annu Rev Cell Biol. 1992;8:133–155. doi: 10.1146/annurev.cb.08.110192.001025. [DOI] [PubMed] [Google Scholar]
  25. Morgan B. A., Johnson W. A., Hirsh J. Regulated splicing produces different forms of dopa decarboxylase in the central nervous system and hypoderm of Drosophila melanogaster. EMBO J. 1986 Dec 1;5(12):3335–3342. doi: 10.1002/j.1460-2075.1986.tb04648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pirrotta V. Vectors for P-mediated transformation in Drosophila. Biotechnology. 1988;10:437–456. doi: 10.1016/b978-0-409-90042-2.50028-3. [DOI] [PubMed] [Google Scholar]
  27. Pyper J. M., Bolen J. B. Identification of a novel neuronal C-SRC exon expressed in human brain. Mol Cell Biol. 1990 May;10(5):2035–2040. doi: 10.1128/mcb.10.5.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rio D. C. Splicing of pre-mRNA: mechanism, regulation and role in development. Curr Opin Genet Dev. 1993 Aug;3(4):574–584. doi: 10.1016/0959-437x(93)90093-5. [DOI] [PubMed] [Google Scholar]
  29. Rosenfeld M. G., Amara S. G., Evans R. M. Alternative RNA processing: determining neuronal phenotype. Science. 1984 Sep 21;225(4668):1315–1320. doi: 10.1126/science.6089345. [DOI] [PubMed] [Google Scholar]
  30. Ryner L. C., Baker B. S. Regulation of doublesex pre-mRNA processing occurs by 3'-splice site activation. Genes Dev. 1991 Nov;5(11):2071–2085. doi: 10.1101/gad.5.11.2071. [DOI] [PubMed] [Google Scholar]
  31. Santoni M. J., Barthels D., Vopper G., Boned A., Goridis C., Wille W. Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. EMBO J. 1989 Feb;8(2):385–392. doi: 10.1002/j.1460-2075.1989.tb03389.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shen J., Beall C. J., Hirsh J. Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock. Mol Cell Biol. 1993 Aug;13(8):4549–4555. doi: 10.1128/mcb.13.8.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Siebel C. W., Rio D. C. Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression. Science. 1990 Jun 8;248(4960):1200–1208. doi: 10.1126/science.2161558. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Stamm S., Zhang M. Q., Marr T. G., Helfman D. M. A sequence compilation and comparison of exons that are alternatively spliced in neurons. Nucleic Acids Res. 1994 May 11;22(9):1515–1526. doi: 10.1093/nar/22.9.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stroeher V. L., Gaiser J. C., Garber R. L. Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions. Mol Cell Biol. 1988 Oct;8(10):4143–4154. doi: 10.1128/mcb.8.10.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tacke R., Goridis C. Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site. Genes Dev. 1991 Aug;5(8):1416–1429. doi: 10.1101/gad.5.8.1416. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Tian M., Maniatis T. Positive control of pre-mRNA splicing in vitro. Science. 1992 Apr 10;256(5054):237–240. doi: 10.1126/science.1566072. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Zachar Z., Chou T. B., Bingham P. M. Evidence that a regulatory gene autoregulates splicing of its transcript. EMBO J. 1987 Dec 20;6(13):4105–4111. doi: 10.1002/j.1460-2075.1987.tb02756.x. [DOI] [PMC free article] [PubMed] [Google Scholar]