Positive and negative intronic regulatory elements control muscle-specific alternative exon splicing of Drosophila myosin heavy chain transcripts (original) (raw)

Abstract

Alternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts is precisely regulated to ensure the expression of specific MHC isoforms required for the distinctive contractile activities of physiologically specialized muscles. We have used transgenic expression analysis in combination with mutagenesis to identify cis-regulatory sequences that are required for muscle-specific splicing of exon 11, which is encoded by five alternative exons that produce alternative "converter" domains in the MHC head. Here, we report the identification of three conserved intronic elements (CIE1, -2, and -3) that control splicing of exon 11e in the indirect flight muscle (IFM). Each of these CIE elements has a distinct function: CIE1 acts as a splice repressor, while CIE2 and CIE3 behave as splice enhancers. These CIE elements function in combination with a nonconsensus splice donor to direct IFM-specific splicing of exon 11e. An additional cis-regulatory element that is essential in coordinating the muscle-specific splicing of other alternative exon 11s is identified. Therefore, multiple interacting intronic and splice donor elements establish the muscle-specific splicing of alternative exon 11s.

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Selected References

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  1. 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]
  2. Chen C. D., Kobayashi R., Helfman D. M. Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev. 1999 Mar 1;13(5):593–606. doi: 10.1101/gad.13.5.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clouet d'Orval B., d'Aubenton-Carafa Y., Brody J. M., Brody E. Determination of an RNA structure involved in splicing inhibition of a muscle-specific exon. J Mol Biol. 1991 Oct 5;221(3):837–856. doi: 10.1016/0022-2836(91)80179-x. [DOI] [PubMed] [Google Scholar]
  4. Dominguez R., Freyzon Y., Trybus K. M., Cohen C. Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state. Cell. 1998 Sep 4;94(5):559–571. doi: 10.1016/s0092-8674(00)81598-6. [DOI] [PubMed] [Google Scholar]
  5. Du C., McGuffin M. E., Dauwalder B., Rabinow L., Mattox W. Protein phosphorylation plays an essential role in the regulation of alternative splicing and sex determination in Drosophila. Mol Cell. 1998 Dec;2(6):741–750. doi: 10.1016/s1097-2765(00)80289-0. [DOI] [PubMed] [Google Scholar]
  6. Dye B. T., Buvoli M., Mayer S. A., Lin C. H., Patton J. G. Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2. RNA. 1998 Dec;4(12):1523–1536. doi: 10.1017/s1355838298980360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. George E. L., Ober M. B., Emerson C. P., Jr Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons. Mol Cell Biol. 1989 Jul;9(7):2957–2974. doi: 10.1128/mcb.9.7.2957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gersappe A., Pintel D. J. CA- and purine-rich elements form a novel bipartite exon enhancer which governs inclusion of the minute virus of mice NS2-specific exon in both singly and doubly spliced mRNAs. Mol Cell Biol. 1999 Jan;19(1):364–375. doi: 10.1128/mcb.19.1.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Hastings G. A., Emerson C. P., Jr Myosin functional domains encoded by alternative exons are expressed in specific thoracic muscles of Drosophila. J Cell Biol. 1991 Jul;114(2):263–276. doi: 10.1083/jcb.114.2.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hodges D., Bernstein S. I. Suboptimal 5' and 3' splice sites regulate alternative splicing of Drosophila melanogaster myosin heavy chain transcripts in vitro. Mech Dev. 1992 May;37(3):127–140. doi: 10.1016/0925-4773(92)90075-u. [DOI] [PubMed] [Google Scholar]
  13. Hodges D., Cripps R. M., O'Connor M. E., Bernstein S. I. The role of evolutionarily conserved sequences in alternative splicing at the 3' end of Drosophila melanogaster myosin heavy chain RNA. Genetics. 1999 Jan;151(1):263–276. doi: 10.1093/genetics/151.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Labourier E., Allemand E., Brand S., Fostier M., Tazi J., Bourbon H. M. Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1. Nucleic Acids Res. 1999 Jun 1;27(11):2377–2386. doi: 10.1093/nar/27.11.2377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Lou H., Helfman D. M., Gagel R. F., Berget S. M. Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon. Mol Cell Biol. 1999 Jan;19(1):78–85. doi: 10.1128/mcb.19.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miedema K., Harhangi H., Mentzel S., Wilbrink M., Akhmanova A., Hooiveld M., Bindels P., Hennig W. Interspecific sequence comparison of the muscle-myosin heavy-chain genes from Drosophila hydei and Drosophila melanogaster. J Mol Evol. 1994 Oct;39(4):357–368. doi: 10.1007/BF00160268. [DOI] [PubMed] [Google Scholar]
  20. Mullen M. P., Smith C. W., Patton J. G., Nadal-Ginard B. Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice. Genes Dev. 1991 Apr;5(4):642–655. doi: 10.1101/gad.5.4.642. [DOI] [PubMed] [Google Scholar]
  21. Nadal-Ginard B., Smith C. W., Patton J. G., Breitbart R. E. Alternative splicing is an efficient mechanism for the generation of protein diversity: contractile protein genes as a model system. Adv Enzyme Regul. 1991;31:261–286. doi: 10.1016/0065-2571(91)90017-g. [DOI] [PubMed] [Google Scholar]
  22. Nagel R. J., Lancaster A. M., Zahler A. M. Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55. RNA. 1998 Jan;4(1):11–23. [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Schaal T. D., Maniatis T. Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences. Mol Cell Biol. 1999 Mar;19(3):1705–1719. doi: 10.1128/mcb.19.3.1705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southby J., Gooding C., Smith C. W. Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons. Mol Cell Biol. 1999 Apr;19(4):2699–2711. doi: 10.1128/mcb.19.4.2699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Standiford D. M., Davis M. B., Sun W., Emerson C. P., Jr Splice-junction elements and intronic sequences regulate alternative splicing of the Drosophila myosin heavy chain gene transcript. Genetics. 1997 Oct;147(2):725–741. doi: 10.1093/genetics/147.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Xiao S. H., Manley J. L. Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2. EMBO J. 1998 Nov 2;17(21):6359–6367. doi: 10.1093/emboj/17.21.6359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xu R., Teng J., Cooper T. A. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. doi: 10.1128/mcb.13.6.3660. [DOI] [PMC free article] [PubMed] [Google Scholar]