Abstract
The mouse c-src gene contains a short neuron-specific exon, N1. To characterize the sequences that regulate N1 splicing, we used a heterologous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Various fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon. These clones were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Several sequences from the N1 exon region induced the splicing of the heterologous exon. The most powerful effect was seen with a sequence from the intron downstream of the N1 exon. This sequence acted as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer was strongest in neuronal LA-N-5 cells but also activated splicing in nonneuronal HEK293 cells. Deletion and linker scanning mutagenesis indicate that the enhancer is made up of multiple smaller elements that must act in combination. One of these elements was identified as the sequence UGCAUG. Three copies of this element can strongly activate splicing of the test exon in LA-N-5 neuroblastoma cells. These component elements of the src splicing enhancer are also apparently involved in the splicing of other short cassette exons.
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- 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]
- 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]
- 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]
- 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]
- Black D. L. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Dirksen W. P., Hampson R. K., Sun Q., Rottman F. M. A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA. J Biol Chem. 1994 Mar 4;269(9):6431–6436. [PubMed] [Google Scholar]
- 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]
- Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Krämer A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem. 1996;65:367–409. doi: 10.1146/annurev.bi.65.070196.002055. [DOI] [PubMed] [Google Scholar]
- 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]
- Lynch K. W., Maniatis T. Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes Dev. 1996 Aug 15;10(16):2089–2101. doi: 10.1101/gad.10.16.2089. [DOI] [PubMed] [Google Scholar]
- Lynch K. W., Maniatis T. Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 1995 Feb 1;9(3):284–293. doi: 10.1101/gad.9.3.284. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Tacke R., Chen Y., Manley J. L. Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1148–1153. doi: 10.1073/pnas.94.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tian H., Kole R. Selection of novel exon recognition elements from a pool of random sequences. Mol Cell Biol. 1995 Nov;15(11):6291–6298. doi: 10.1128/mcb.15.11.6291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]