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Mol Cell Biol. 1997 August; 17(8): 4667–4676.
PMCID: PMC232319

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


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.

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Selected References

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  • Adams MD, Rudner DZ, Rio DC. Biochemistry and regulation of pre-mRNA splicing. Curr Opin Cell Biol. 1996 Jun;8(3):331–339. [PubMed]
  • Amendt BA, Si ZH, Stoltzfus CM. 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. [PMC free article] [PubMed]
  • Balvay L, Libri D, Gallego M, Fiszman MY. 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. [PMC free article] [PubMed]
  • Bell LR, Horabin JI, Schedl P, Cline TW. Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell. 1991 Apr 19;65(2):229–239. [PubMed]
  • Berget SM. Exon recognition in vertebrate splicing. J Biol Chem. 1995 Feb 10;270(6):2411–2414. [PubMed]
  • Black DL. 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. [PubMed]
  • Black DL. 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. [PubMed]
  • Black DL. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PubMed]
  • Boggs RT, Gregor P, Idriss S, Belote JM, 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. [PubMed]
  • Burd CG, 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. [PubMed]
  • 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. [PubMed]
  • Caputi M, Casari G, Guenzi S, Tagliabue R, Sidoli A, Melo CA, Baralle FE. 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. [PMC free article] [PubMed]
  • Chabot B. Directing alternative splicing: cast and scenarios. Trends Genet. 1996 Nov;12(11):472–478. [PubMed]
  • Chan RC, Black DL. Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro. Mol Cell Biol. 1995 Nov;15(11):6377–6385. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Deshpande G, Samuels ME, Schedl PD. Sex-lethal interacts with splicing factors in vitro and in vivo. Mol Cell Biol. 1996 Sep;16(9):5036–5047. [PMC free article] [PubMed]
  • Dominski Z, Kole R. Selection of splice sites in pre-mRNAs with short internal exons. Mol Cell Biol. 1991 Dec;11(12):6075–6083. [PMC free article] [PubMed]
  • Estes PA, Cooke NE, Liebhaber SA. 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]
  • García-Blanco MA, Jamison SF, Sharp PA. 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. [PubMed]
  • Ghetti A, Piñol-Roma S, Michael WM, 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. [PMC free article] [PubMed]
  • Gooding C, Roberts GC, Moreau G, Nadal-Ginard B, Smith CW. 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. [PubMed]
  • Graham IR, Hamshere M, Eperon IC. Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences. Mol Cell Biol. 1992 Sep;12(9):3872–3882. [PMC free article] [PubMed]
  • Guo W, Mulligan GJ, Wormsley S, Helfman DM. 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. [PubMed]
  • Helfman DM, Ricci WM, Finn LA. Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo. Genes Dev. 1988 Dec;2(12A):1627–1638. [PubMed]
  • Horabin JI, 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PubMed]
  • Levy JB, Dorai T, Wang LH, Brugge JS. 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. [PMC free article] [PubMed]
  • Libri D, Balvay L, Fiszman MY. 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. [PMC free article] [PubMed]
  • Libri D, Piseri A, Fiszman MY. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure. Science. 1991 Jun 28;252(5014):1842–1845. [PubMed]
  • Lin CH, Patton JG. Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA. 1995 May;1(3):234–245. [PubMed]
  • Lou H, Gagel RF, Berget SM. An intron enhancer recognized by splicing factors activates polyadenylation. Genes Dev. 1996 Jan 15;10(2):208–219. [PubMed]
  • 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. [PubMed]
  • Mayeda A, Munroe SH, Cáceres JF, Krainer AR. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. 1994 Nov 15;13(22):5483–5495. [PubMed]
  • McKeown M. Sex differentiation: the role of alternative splicing. Curr Opin Genet Dev. 1992 Apr;2(2):299–303. [PubMed]
  • Min H, Chan RC, Black DL. The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event. Genes Dev. 1995 Nov 1;9(21):2659–2671. [PubMed]
  • Mulligan GJ, Guo W, Wormsley S, Helfman DM. 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]
  • Nemeroff ME, Utans U, Krämer A, Krug RM. 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. [PMC free article] [PubMed]
  • Norton PA, Hynes RO. 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. [PubMed]
  • O'Reilly MM, McNally MT, Beemon KL. 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. [PubMed]
  • Patton JG, Mayer SA, 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. [PubMed]
  • Patton JG, Porro EB, 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. [PubMed]
  • Reed R. Initial splice-site recognition and pairing during pre-mRNA splicing. Curr Opin Genet Dev. 1996 Apr;6(2):215–220. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Samuels ME, Bopp D, Colvin RA, Roscigno RF, Garcia-Blanco MA, Schedl P. RNA binding by Sxl proteins in vitro and in vivo. Mol Cell Biol. 1994 Jul;14(7):4975–4990. [PMC free article] [PubMed]
  • Siebel CW, Fresco LD, Rio DC. 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. [PubMed]
  • Singh R, Valcárcel J, Green MR. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. [PubMed]
  • Sirand-Pugnet P, Durosay P, Clouet d'Orval BC, 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. [PubMed]
  • Sosnowski BA, Belote JM, 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Swanson MS, 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. [PMC free article] [PubMed]
  • Swanson MS, 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. [PubMed]
  • Valcárcel J, Singh R, Zamore PD, Green MR. 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. [PubMed]

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