An Intronic Polypyrimidine-rich Element Downstream of the Donor Site Modulates Cystic Fibrosis Transmembrane Conductance Regulator Exon 9 Alternative Splicing
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313439200
ISSN1083-351X
AutoresElisabetta Zuccato, Emanuele Buratti, Cristiana Stuani, Francisco E. Baralle, Franco Pagani,
Tópico(s)Cystic Fibrosis Research Advances
ResumoTwo intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041–21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774–1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5′-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5′-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5′-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3′-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5′-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5′-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3′-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1. Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041–21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774–1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5′-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5′-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5′-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3′-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5′-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5′-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3′-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1. Cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ISS, intronic splicing silencer; SR, Ser/Arg-rich; PTB, polypyrimidine tract-binding protein; ss, splice site; snRNP, small nuclear ribonucleoprotein; PCE, polypyrimidine-rich controlling element; PY, polypyrimidine-rich; RT, reverse transcription; snRNA, small nuclear RNA. is the most common autosomal recessive disorder in Caucasians, and it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1Welsh M. Tsui L.-C. Boat T. Beaudet A. Hill M.G. The Metabolic Bases of Inherited Disease. McGraw Hill, New York1995: 3799-3876Google Scholar). CFTR mutations can also be associated with non-classical forms of CF in which the disease shows a tissue-specific variability such as congenital bilateral absence of vas deference and idiopathic pancreatitis (2Cohn J.A. Friedman K.J. Noone P.G. Knowles M.R. Silverman L.M. Jowell P.S. N. Engl. J. Med. 1998; 339: 653-658Crossref PubMed Scopus (807) Google Scholar). In some cases, the phenotypic variability has been associated with a variable proportion of aberrant CFTR exon 9 skipping, which produces a nonfunctional protein (3Chu C.S. Trapnell B.C. Curristin S. Cutting G.R. Crystal R.G. Nat. Genet. 1993; 3: 151-156Crossref PubMed Scopus (441) Google Scholar, 4Chillon M. Casals T. Mercier B. Bassas L. Lissens W. Silber S. Romey M.C. Ruiz-Romero J. Verlingue C. Claustres M. Nunes V. Férec C. Estivill X. N. Engl. J. Med. 1995; 332: 1475-1480Crossref PubMed Scopus (818) Google Scholar, 5Larriba S. Bassas L. Gimenez J. Ramos M.D. Segura A. Nunes V. Estivill X. Casals T. Hum. Mol. Genet. 1998; 7: 1739-1743Crossref PubMed Scopus (64) Google Scholar, 6Mak V. Jarvi K.A. Zielenski J. Durie P. Tsui L.C. Hum. Mol. Genet. 1997; 6: 2099-2107Crossref PubMed Scopus (93) Google Scholar, 7Cuppens H. Lin W. Jaspers M. Costes B. Teng H. Vankeerberghen A. Jorissen M. Droogmans G. Reynaert I. Goossens M. Nilius B. Cassiman J.J. J. Clin. Investig. 1998; 101: 487-496Crossref PubMed Scopus (355) Google Scholar) and can be modulated by splicing factors. Extensive studies on CFTR exon 9 alternative splicing have provided a paradigmatic example of the complexity of its regulation and, in this case, the possibility of aberrant exon skipping that leads to pathological consequences. Several cis-acting elements and trans-acting factors have been identified to modulate CFTR exon 9 alternative splicing. The cis-acting elements described so far are the (TG)m(T)n polymorphic locus at 3′-end of IVS8 (3Chu C.S. Trapnell B.C. Curristin S. Cutting G.R. Crystal R.G. Nat. Genet. 1993; 3: 151-156Crossref PubMed Scopus (441) Google Scholar), the juxtaposed enhancer and silencer exonic elements (8Pagani F. Buratti E. Stuani C. Baralle F.E. J. Biol. Chem. 2003; 278: 26580-26588Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), and the intronic splicing silencer (ISS). Several in vitro and in vivo studies have shown that a high number of TG repeats and a low number of T tracts in the TGmTn locus induce exon skipping. In intron 9, the ISS element is located 75 bp downstream of the weak 5′-splice site (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In our previous studies, we identified TDP43 and members of the Ser/Arg-rich (SR) protein family as inhibitors of CFTR exon 9 alternative splicing via their interaction with the TG tract and the ISS, respectively (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 10Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (499) Google Scholar); interestingly, the expression level of these proteins is higher in the mostly affected CF tissues (10Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (499) Google Scholar). No enhancing splicing factor has been identified so far to activate CFTR exon 9 inclusion. Its role could be important to counteract splicing inhibition and to modulate the tissue-specific phenotypic expression in non-classical CF forms. TIA-1 and polypyrimidine tract-binding protein (PTB) are two splicing factors involved in the regulation of alternative splicing. Both recognize intronic polypyrimidine-rich sequences, mainly located in introns, and promote exon inclusion or skipping, respectively. TIA-1 binding to polypyrimidine-rich sequences downstream of the 5′-splice site (ss) is involved in the recognition of exons that contain weak 5′-splice sites. Sequence analysis of TIA-1, initially identified as an apoptotic protein (11Tian Q. Streuli M. Saito H. Schlossman S.F. Anderson P. Cell. 1991; 67: 629-639Abstract Full Text PDF PubMed Scopus (335) Google Scholar), revealed that it contains three RNA recognition motifs, and its amino acid sequence shows close homology to the Saccharomyces cerevisiae Nam8p protein, a U1 small nuclear ribonucleoprotein (snRNP) complex component (12Gottschalk A. Tang J. Puig O. Salgado J. Neubauer G. Colot H.V. Mann M. Seraphin B. Rosbash M. Luhrmann R. Fabrizio P. RNA (N. Y.). 1998; 4: 374-393PubMed Google Scholar). In vitro selection/amplification assays have shown a preference for TIA-1 binding to RNAs containing short stretches of uridines, although a core binding sequence has not been recognized (13Dember L.M. Kim N.D. Liu K.Q. Anderson P. J. Biol. Chem. 1996; 271: 2783-2788Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Acting on weak 5′-splice sites followed by U-rich stretches, TIA-1 modulates the alternative splicing of Drosophila msl-2 and human fas receptor pre-mRNAs (14Forch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcarcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The TIA-1 effect may be critical for U1 snRNP recruitment and stabilization to these weak 5′-splice sites. Interaction between TIA-1 and U1-C, one of the three U1-specific polypeptides (15Forch P. Puig O. Martinez C. Seraphin B. Valcarcel J. EMBO J. 2002; 21: 6882-6892Crossref PubMed Scopus (182) Google Scholar), stabilizes the association of both proteins and consequently of U1 snRNP with the adjacent 5′-ss followed by the U-rich stretch. In one case, TIA-1 in vivo depletion resulted in a different choice of a wild type normal 3′-ss partner, but the mechanism involved is not known (14Forch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcarcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). U-rich polypyrimidine tracts can also interact with PTB. PTB is a well characterized splicing factor involved in repression of the cell-specific splicing pattern in inappropriate cell types (16Wagner E.J. Garcia-Blanco M.A. Mol. Cell. Biol. 2001; 21: 3281-3288Crossref PubMed Scopus (304) Google Scholar). Multiple intronic PTB-binding sites distributed on the entire length of pre-mRNAs have been found to mediate splicing repression in several alternatively spliced mRNAs (17Carstens R.P. McKeehan W.L. Garcia-Blanco M.A. Mol. Cell. Biol. 1998; 18: 2205-2217Crossref PubMed Scopus (85) Google Scholar, 18Gooding C. Roberts G.C. Smith C.W. RNA (N. Y.). 1998; 4: 85-100PubMed Google Scholar, 19Southby J. Gooding C. Smith C.W. Mol. Cell. Biol. 1999; 19: 2699-2711Crossref PubMed Google Scholar, 20Norton P.A. Nucleic Acids Res. 1994; 22: 3854-3860Crossref PubMed Scopus (72) Google Scholar, 21Mulligan G.J. Guo W. Wormsley S. Helfman D.M. J. Biol. Chem. 1992; 267: 25480-25487Abstract Full Text PDF PubMed Google Scholar, 22Chan R.C. Black D.L. Mol. Cell. Biol. 1997; 17: 4667-4676Crossref PubMed Google Scholar). The ability of PTB to multimerize led to the suggestion that it establishes a "zone of silencing" within a pre-mRNA, preventing splice site usage and that, in this manner, it mediates exon silencing (16Wagner E.J. Garcia-Blanco M.A. Mol. Cell. Biol. 2001; 21: 3281-3288Crossref PubMed Scopus (304) Google Scholar, 23Wagner E.J. Garcia-Blanco M.A. Mol. Cell. 2002; 10: 943-949Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In this study, we show that the region between the weak 5′-ss of CFTR exon 9 and the ISS has an important regulatory role in alternative splicing. This polypyrimidine-rich controlling element (PCE) contains a peculiar arrangement of three polypyrimidine-rich (PY) motifs. TIA-1 binding to the PCE increased exon 9 inclusion, possibly stabilizing U1 snRNP interaction with pre-mRNA. In contrast, PTB showed an opposite effect and caused an increase in exon 9 skipping. In addition, we also report that TIA-1 induced the selection of a cryptic exonic 3′-ss independently of its facilitating role in 5′-ss recognition mediated by U1 snRNP. Taken together, these data increase the number of factors involved in the complex regulation of CFTR exon 9 alternative splicing and for the first time show the involvement of a splicing factor with enhancing activity in CFTR exon 9 recognition. Plasmid Construction—The wild-type constructs carrying variable combinations of (TG)m(T)n repeats at the polymorphic locus and hCF-Δint2 have been described previously (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The mutations introduced in the PY motifs were obtained by PCR with different oligonucleotide sets using templates with different mutations inserted. The resulting products were digested with BamHI/KpnI and cloned into the CFTR intron 9 sequence contained in the NdeI insert of the modified pBluescript plasmid (pBSCFNde) described previously (24Niksic M. Romano M. Buratti E. Pagani F. Baralle F.E. Hum. Mol. Genet. 1999; 8: 2339-2349Crossref PubMed Scopus (129) Google Scholar). The mutagenized NdeI fragment was then subcloned in the NdeI-digested TG11T5 minigene. The BamHI/KpnI cassettes were created by PCR-mediated site-directed mutagenesis using different primers for each cassette: M1, CF9junAdir (5′-ctggatccactggagcaggcaaggtagttcatttg-3′); M2, CFA/3-2bsdir (5′-ctggatccactggagcaggcaaggtagttcttttgttcatc-3′); M1,2, the same primer as M1, but on the M2 template; M2A, CF9/2bsmut (5′-ctggatccactggagcaggcaaggtagttcttttgtaaatc-3′); and M1,2A, CF9/1,2bsmut (5′-ctggatccactggagcaggcaaggtagtgaatttgtaaatc-3′). For all amplifications, the reverse primer used was UNI REV (5′-ggaaacagctatgaccatg-3′). For M3, a two-step overlap extension method was used. For the first amplification, the two sets of primers used were as follows: hCFex9/in9dir (5′-ctggatccactggagcaggcaaggtagttcttttg-3′) and ΔYrev (5′-caaactgcaggacaccaaattaagttcttaat-3′); and ΔYdir (5′-gtgtcctgcagtttgtagtgctggaaggtat-5′) and UNI REV. For the second amplification, the hCFex9/in9dir and UNI REV set was used. For M1,2,3, CF9/1,2bsmut (5′-ctggatccactggagcaggcaaggtagtgaatttgtaaatc-3′) and UNI REV on the ΔM3 template were used. A derivative of pHU1 (25Pagani F. Buratti E. Stuani C. Bendix R. Dork T. Baralle F.E. Nat. Genet. 2002; 30: 426-429Crossref PubMed Scopus (189) Google Scholar) was used to prepare EX9U1-5′-ss. This variant was created as described (25Pagani F. Buratti E. Stuani C. Bendix R. Dork T. Baralle F.E. Nat. Genet. 2002; 30: 426-429Crossref PubMed Scopus (189) Google Scholar) using oligonucleotides EX9U1-5′-ssDIR (5′-gatctcagaactacctggcaggggagataccat-3′) and EX9U1-5′-ssREV (5′-gatcatggtatctcccctgccaggtagttctga-3′). Analysis of Hybrid Minigene Expression—Transient transfection of Hep3B cells, RNA extraction, reverse transcription (RT)-PCR, and quantitation of PCR products were done as described previously (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The α2 and B2 primers have been described previously (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The sequence of the cry2 primer is (5′-ggaggaacagcttgcctgctcc-3′). The pHI plasmid was kindly provided by Dr. M. G. Romanelli. The pTIA-1 expression plasmid was kindly provided by Dr. R. Breathnach. If not specified otherwise, 1 μg of pHI or pTIA-1 plasmid was used in cotransfection experiments. UV Cross-linking Assay—The T7SW Eco competitor was prepared by cutting and religating the pBSCFNde construct with EcoRI and KpnI enzymes. The 3′-h3′int competitor was prepared by cutting and religating the pBSCFNde construct with SacI and BamHI enzymes. The pBIND competitor was prepared by cloning the EcoRI/BamHI fragment of exon 9 into pBSSK. The competitors in intron 9 that span the first 77 nucleotides were prepared directly by cloning the PCR product (direct primer, hCFex9/in9dir; and reverse primer, IN9Hind/Kpn (5′-ggaagcttccaaaagcttccagcac-3′)) digested with BamHI/HindIII into pBSSK. The pEND competitor was prepared digesting the ΔM3 PCR product with PstI/KpnI and cloning the insert into pBSSK. Each competitor was linearized and used for in vitro transcription and UV cross-linking assays as described previously (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Immunoprecipitation Experiments—Polyclonal antiserum against PTB was obtained by immunizing a New Zealand White 3-month-old rabbit according to standard protocols. PTB immunoprecipitation experiments were done as described previously (10Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (499) Google Scholar). TIA-1 immunoprecipitation experiments were performed as described (26Del Gatto-Konczak F. Bourgeois C.F. Le Guiner C. Kister L. Gesnel M.C. Stevenin J. Breathnach R. Mol. Cell. Biol. 2000; 20: 6287-6299Crossref PubMed Scopus (169) Google Scholar) using a commercially available anti-TIA-1 polyclonal antibody (Santa Cruz Biotechnology). Pull-down Assay—The unlabeled RNA probes were prepared as described previously (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and placed in a 400-μl reaction mixture containing 0.1 m NaOAc (pH 5.0) and 5 mm sodium metaperiodate (Sigma). Reaction mixtures were incubated for 1 h in the dark at room temperature. The RNA was ethanol-precipitated and resuspended in 100 μl of 0.1 m NaOAc (pH 5.0). Then, 300 μl of a 50% adipic acid dihydrazideagarose bead slurry (Sigma) were washed four times with 10 ml of 0.1 m NaOAc (pH 5.0) and pelleted after each wash at 3000 rpm for 3 min. After the final wash, 300 μl of 0.1 m NaOAc (pH 5.0) were added to the beads. The slurry was then mixed with the periodate-treated RNA and incubated for 12 h at 4 °C on a rotator. The beads with the bound RNA were pelleted and washed three times with 2 ml of 2 m NaCl and three times with 3 ml of buffer A (20 mm HEPES-KOH (pH 7), 6.5% (v/v) glycerol, 0.1 m KCl, 0.2 mm EDTA, and 0.5 mm dithiothreitol). They were incubated with 0.6 mg of HeLa cell nuclear extract for 20 min at 30 °C in a final volume of 650 μl, pelleted by centrifugation at 1000 rpm for 3 min, and washed five times with 5 ml of buffer A containing 4 mm MgCl2. After the final centrifugation, 60 μl of SDS-PAGE sample buffer were added to the beads and heated for 5 min at 90 °C before loading onto a 10% SDS-polyacrylamide gel. PY Motifs near the CFTR Exon 9 5′-ss Are Intronic Splicing Enhancers—The intronic region between the 5′-ss of CFTR exon 9 and the previously reported ISS contains three distinct pyrimidine-rich elements, PY1, PY2, and PY3 (Fig. 1B). PY1 and PY2 are separated only by a purine residue and together span 13 nucleotides close to the exon/intron junction. The PY3 element is located 30 nucleotides downstream of PY2 and is composed of 15 contiguous pyrimidine residues (Fig. 1B). We prepared CFTR exon 9 hybrid minigenes containing mutations in the pyrimidine-rich elements as shown in Fig. 1B. Hybrid minigenes were transfected in Hep3B cells, and the pattern of splicing was analyzed by RT-PCR amplification using specific primers. Fig. 1C shows the effects of single and multiple mutations on the efficiency of splicing. The control wild-type hCFTG11T5 construct (lane 1) resulted in 65 ± 5% exon 9 inclusion, as previously reported (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Using the M1 and M3 constructs, with mutations in PY1 and PY3, the level of exon 9 inclusion was ∼60%, i.e. not significantly different from TG11T5 (Fig. 1C, lanes 2 and 7). Disruption of the PY2 motif in M2 (TCTT motif in PY2 changed to TCAT) and M2A (TCTT motif changed to AAAT) produced 35 ± 3 and 37 ± 4% exon inclusion, respectively (Fig. 1C, lanes 3 and 5). Significantly lower levels of exon 9 inclusion were observed in the double mutants M1,2 and M1,2A, corresponding to 22 ± 4 and 23 ± 2%, respectively (Fig. 1C, lanes 4 and 6), indicating that mutations in PY1 contribute to splicing regulation when associated with mutations in PY2 (Fig. 1C, compare lanes 3 and 4). The disruption of all three PY motifs (M1,2,3) resulted in the lowest level of exon 9 inclusion (13 ± 2%) (Fig. 1C, lane 8), which was also significantly different compared with the M1,2 and M1,2A mutants, suggesting a combined effect of the three PY motifs. Our previous work also showed that an exon 9 cryptic band originated from the recognition of a cryptic 3′-ss in exon 9 (10Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (499) Google Scholar). Using the same construct (hcFTG11T5), the percentage of this cryptic band was very low and did not change significantly upon comparison of the wild-type and mutant constructs. These results indicate that the intronic sequence between the 5′-ss and the ISS, which we have named PCE for polypyrimidine-rich controlling element, constitutes a new intronic splicing regulatory element involved in the modulation of CFTR exon 9 alternative splicing. Functional Relationship between the (TG)m(T)n Locus, ISS, and PCE—Previous studies have shown that exon 9 inclusion is modulated by the polymorphic (TG)m(T)n locus near the 3′-ss and by the ISS in intron 9 (3Chu C.S. Trapnell B.C. Curristin S. Cutting G.R. Crystal R.G. Nat. Genet. 1993; 3: 151-156Crossref PubMed Scopus (441) Google Scholar, 9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). An increase in the length of the TG tract or a reduction of the U-rich stretch induces exon 9 skipping. In addition, a high number of TG repeats (>13) and a low number of T repeats (<5) induces a significant activation of a cryptic 3′-ss in exon 9. We have now evaluated the enhancing effect of the PCE in relation to the ISS and to the composition of the polymorphic (TG)m(T)n locus. Mutations in the three PY motifs in the PCE were evaluated in the context of the TG11T7 and TG13T5 variants of intron 8 or in combination with deletion of the ISS. The levels of exon 9 inclusion in the wild-type TG11T7 and TG13T5 minigenes were 90 and 40%, respectively (Fig. 1D, lanes 1 and 3). In comparison with these constructs, the TG11T7/M1,2,3 and TG13T5/M1,2,3 minigenes resulted in reduced exon 9 inclusion (80 and 7%, respectively) (Fig. 1D, lanes 2 and 4). The TG13T5 minigenes showed, as expected, usage of the cryptic 3′-ss, but the percentage of inclusion was not significantly affected in the PCE mutants (see additional experiments in Fig. 5). Deletion of the ISS from the wild-type minigene (hCFΔISS) resulted in 95% exon inclusion (Fig. 1D, lane 5) because of the absence of the ISS inhibitory element, as previously reported (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In comparison with hCFΔISS, mutations in all of the PY motifs (M1,2,3ΔISS) reduced the level of exon inclusion to 55% (Fig. 1D, lane 8). Mutation in PY1 (M1ΔISS) or in both PY1 and PY2 (M1,2ΔISS) produced 90 and 75% exon inclusion, respectively (Fig. 1D, lanes 6 and 7). These results indicate that the enhancing effect of the PCE is modulated by differences in the (TG)m(T)n locus and by the ISS element. Thus, the PCE represents a new intronic element that, along with the ISS in intron 9 and the (TG)m(T)n element in intron 8, modulates CFTR exon 9 alternative splicing. This new intronic element could affect the efficiency of recognition of the upstream weak 5′-ss through binding of a specific splicing factor to the PY motifs. Role of the Polypyrimidine-binding Proteins TIA-1 and PTB in CFTR Exon 9 Alternative Splicing—To study the influence of TIA-1 and PTB on CFTR exon 9 alternative splicing, we performed transient cotransfection experiments. Hep3B cells were transfected with the TG11T5 minigene alone or with increasing amounts of TIA-1 or PTB expression vector. TIA-1 expression induced a dose-dependent increase in exon 9 inclusion (Fig. 2A). In fact, in comparison with the basal level of exon 9 inclusion obtained using the TG11T5 minigene (65%), TIA-1 overexpression increased inclusion to ∼90% at the highest vector concentrations (Fig. 2A). In contrast, cotransfection with increasing amounts of the PTB expression vector (pHI) resulted in dose-dependent inhibition of exon 9 splicing. It is interesting to note that TIA-1 is the only splicing factor identified so far that stimulates CFTR exon 9 inclusion. In fact, heterogeneous nuclear ribonucleoprotein A1, TDP43, PTB, and several other SR proteins induce CFTR exon 9 skipping (9Pagani F. Buratti E. Stuani C. Romano M. Zuccato E. Niksic M. Giglio L. Faraguna D. Baralle F.E. J. Biol. Chem. 2000; 275: 21041-21047Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 10Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (499) Google Scholar). To evaluate the role of the multiple PY motifs, we studied the effect of TIA-1 and PTB overexpression using several mutant PCE minigenes (Fig. 2C). The enhancing effect of TIA-1 was observed in the case of the M1, M2, and M2A mutants and the double mutants M1,2 and M1,2A, whereas a lower response was observed with the M3 mutant (Fig. 2C). In contrast, TIA-1 overexpression did not induce exon 9 inclusion with the M1,2,3 minigene, in which all of the PY motifs of the PCE had been mutated (Fig. 2C). This indicates that the effect of TIA-1 on CFTR exon 9 splicing requires mainly the contribution of PY3. However, the complete absence of any response to TIA-1 overexpression in the M1,2,3 minigene suggests the possible additional contribution of the other polypyrimidine-rich sequences of PCE. These results also establish that TIA-1 increases CFTR exon 9 inclusion in a PCE-dependent manner. In contrast, overexpression of PTB reduced exon 9 inclusion with all of the minigenes (Fig. 2D). The percentage of exon 9 inclusion was ∼33–35% with the wild-type, M1, and M3 minigenes and was almost 10–12% with the other mutants. Interestingly, the M1,2,3 minigene was sensitive to PTB overexpression, and the level of exon 9 inclusion was reduced to ∼4% (Fig. 2D). These results are in agreement with the general splicing inhibitory role of PTB, mediated by binding to multiple exonic and intronic sequences (18Gooding C. Roberts G.C. Smith C.W. RNA (N. Y.). 1998; 4: 85-100PubMed Google Scholar, 19Southby J. Gooding C. Smith C.W. Mol. Cell. Biol. 1999; 19: 2699-2711Crossref PubMed Google Scholar, 27Chou M.Y. Underwood J.G. Nikolic J. Luu M.H. Black D.L. Mol. Cell. 2000; 5: 949-957Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). TIA-1 and PTB Binding to the PCE—To investigate the ability of TIA-1 and PTB to bind to the PCE in intron 9, different in vitro transcribed, [32P]UTP-labeled RNA probes (Fig. 3A) were incubated with HeLa nuclear extracts, followed by UV cross-linking. The samples were then immunoprecipitated with specific anti-TIA-1 or anti-PTB polyclonal antibodies. The RNAs used correspond to the last 10 bases of exon 9 and extend down to the ISS element in intron 9. The anti-TIA-1 antibody immunoprecipitated a double band at 40–44 kDa when the wild-type PCE RNA was used (Fig. 3B, lane 2). This doublet corresponds to the previously reported isoforms of this splicing factor (28Kawakami A. Tian Q. Duan X. Streuli M. Schlossman S.F. Anderson P. Proc. Natl.
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