Portal de Periódicos da CAPES

Missense, Nonsense, and Neutral Mutations Define Juxtaposed Regulatory Elements of Splicing in Cystic Fibrosis Transmembrane Regulator Exon 9

2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês

10.1074/jbc.m212813200

1083-351X

Franco Pagani, Emanuele Buratti, Cristiana Stuani, Francisco E. Baralle,

RNA Interference and Gene Delivery

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3′ portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms. Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3′ portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms. The correct definition of exonic and intronic sequences includes not only the recognition of discrete elements at the 3′ and 5′ splice sites, the polypyrimidine tract, and the branch site, but also the contribution of less well defined exonic cis-acting elements, which may influence the use of the flanking splice sites. Exonic sequences have been shown to modulate the splicing efficiency, and single base changes in these sequences may cause pathological splicing events by inducing exon skipping or exon inclusion (1Dietz H.C. Valle D. Francomano C.A. Kendzior Jr., R.J. Pyeritz R.E. Cutting G.R. Science. 1993; 259: 680-683Crossref PubMed Scopus (359) Google Scholar, 2Shiga N. Takeshima Y. Sakamoto H. Inoue K. Yokota Y. Yokoyama M. Matsuo M. J. Clin. Invest. 1997; 100: 2204-2210Crossref PubMed Scopus (132) Google Scholar, 3Lorson C.L. Hahnen E. Androphy E.J. Wirth B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6307-6311Crossref PubMed Scopus (1135) Google Scholar, 4D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (415) Google Scholar). Several mechanisms have been proposed to explain the splicing regulation mediated by these exonic sequences. In some cases, the creation of nonsense mutations has been shown to induce exon skipping, leading to the proposal of a putative nuclear reading frame scanning mechanism for nonsense codons and consequent elimination of the exons (1Dietz H.C. Valle D. Francomano C.A. Kendzior Jr., R.J. Pyeritz R.E. Cutting G.R. Science. 1993; 259: 680-683Crossref PubMed Scopus (359) Google Scholar, 5Dietz H.C. Kendzior Jr., R.J. Nat. Genet. 1994; 8: 183-188Crossref PubMed Scopus (132) Google Scholar, 6Aoufouchi S. Yelamos J. Milstein C. Cell. 1996; 85: 415-422Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Lozano F. Maertzdorf B. Pannell R. Milstein C. EMBO J. 1994; 13: 4617-4622Crossref PubMed Scopus (82) Google Scholar, 8Gersappe A. Pintel D.J. Mol. Cell. Biol. 1999; 19: 1640-1650Crossref PubMed Scopus (32) Google Scholar, 9Li B. Wachtel C. Miriami E. Yahalom G. Friedlander G. Sharon G. Sperling R. Sperling J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5277-5282Crossref PubMed Scopus (51) Google Scholar). In addition, the well established nonsense-mediated decay of mRNAs containing nonsense codons (10Hentze M.W. Kulozik A.E. Cell. 1999; 96: 307-310Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar, 11Maquat L.E. Carmichael G.G. Cell. 2001; 104: 173-176Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) can also be involved in selective degradation of nonsense alternatively spliced mRNAs. However, not all mRNAs are subjected to nonsense-mediated decay (12Romao L. Inacio A. Santos S. Avila M. Faustino P. Pacheco P. Lavinha J. Blood. 2000; 96: 2895-2901Crossref PubMed Google Scholar, 13Pitts S.A. Kullar H.S. Stankovic T. Stewart G.S. Last J.I. Bedenham T. Armstrong S.J. Piane M. Chessa L. Taylor A.M. Byrd P.J. Hum. Mol. Genet. 2001; 10: 1155-1162Crossref PubMed Scopus (51) Google Scholar).Skipping of constitutive exons may also occur for missense and silent mutations. In this case, disruption of exonic splicing enhancer or the creation of exonic splicing silencer has been considered an alternative mechanism to explain the exon skipping phenotype (2Shiga N. Takeshima Y. Sakamoto H. Inoue K. Yokota Y. Yokoyama M. Matsuo M. J. Clin. Invest. 1997; 100: 2204-2210Crossref PubMed Scopus (132) Google Scholar, 10Hentze M.W. Kulozik A.E. Cell. 1999; 96: 307-310Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar, 14Cooper T.A. Mattox W. Am. J. Hum. Genet. 1997; 61: 259-266Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 15Liu H.X. Cartegni L. Zhang M.Q. Krainer A.R. Nat. Genet. 2001; 27: 55-58Crossref PubMed Google Scholar). Some exonic splicing enhancers in pre-mRNAs interact with serine/arginine-rich proteins (SR proteins) 1The abbreviations used are: SR protein, serine/arginine-rich protein; CERES, composite exonic regulatory element of splicing; CF, cystic fibrosis; hCF, human CF; CFTR, cystic fibrosis transmembrane regulator; SF2/ASF, splicing factor 2; hnRNP, heterogeneous ribonucleoprotein; hnRNPH, heterogeneous ribonucleoprotein H; SELEX, systematic evolution of ligands by exponential enrichment; RT, reverse transcription; WT, wild type.1The abbreviations used are: SR protein, serine/arginine-rich protein; CERES, composite exonic regulatory element of splicing; CF, cystic fibrosis; hCF, human CF; CFTR, cystic fibrosis transmembrane regulator; SF2/ASF, splicing factor 2; hnRNP, heterogeneous ribonucleoprotein; hnRNPH, heterogeneous ribonucleoprotein H; SELEX, systematic evolution of ligands by exponential enrichment; RT, reverse transcription; WT, wild type. (16Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (600) Google Scholar). SR proteins are essential splicing factors required for both constitutive and alternative splicing, and changes in their relative concentrations with respect to antagonistic splicing factors have been found to affect splice site selection (17Caceres J.F. Stamm S. Helfman D.M. Krainer A.R. Science. 1994; 265: 1706-1709Crossref PubMed Scopus (561) Google Scholar). Functional systematic evolution of ligands by exponential enrichment (SELEX) strategies have identified highly degenerate consensus sequences binding to SR proteins. These sites are more represented in exons than in introns (18Liu H.X. Zhang M. Krainer A.R. Genes Dev. 1998; 12: 1998-2012Crossref PubMed Scopus (415) Google Scholar, 19Liu H.X. Chew S.L. Cartegni L. Zhang M.Q. Krainer A.R. Mol. Cell. Biol. 2000; 20: 1063-1071Crossref PubMed Scopus (181) Google Scholar). In the BRCA1 and SMN genes, changes in SR protein score motifs derived from these SELEX experiments at exonic splicing enhancer sites have been shown to correlate with the efficiency of splicing (15Liu H.X. Cartegni L. Zhang M.Q. Krainer A.R. Nat. Genet. 2001; 27: 55-58Crossref PubMed Google Scholar, 20Cartegni L. Krainer A.R. Nat. Genet. 2002; 30: 377-384Crossref PubMed Scopus (570) Google Scholar). This has led to the suggestion that SR protein score motif analysis might represent a useful tool to identify general controlling elements of splicing efficiency. On the other hand, exonic splicing silencer elements have always been classically separated from enhancer sequences and may interact with negative regulators, which often belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. In particular, binding of hnRNPH at G-rich sequences has been recently found to exert a strong inhibitory splicing effect in the rat β-tropomyosin gene, in the Rous sarcoma virus, and in human immunodeficiency virus-1 (21Chen C.D. Kobayashi R. Helfman D.M. Genes Dev. 1999; 13: 593-606Crossref PubMed Scopus (167) Google Scholar, 22Fogel B.L. McNally M.T. J. Biol. Chem. 2000; 275: 32371-32378Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 23Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 24Caputi M. Zahler A.M. J. Biol. Chem. 2001; 276: 43850-43859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar).The CFTR exon 9 alternative splicing represents an interesting model to evaluate the contribution of exonic sequences in normal and pathologic pre-mRNA processing. Exon skipping produces a nonfunctional CFTR protein, and alternative splicing of this exon has been associated with monosymptomatic forms of cystic fibrosis (CF). The cis-acting elements so far identified include the polymorphic region at the 3′-end of intron 8 and the intronic splicing silencer in intron 9. The polymorphic locus contains a variable number of dinucleotide TG (from 9 to 13) followed by a T repeat (T5, T7, or T9), and a high number of TG repeats and a low number of T tract induce exon skipping (25Niksic M. Romano M. Buratti E. Pagani F. Baralle F.E. Hum. Mol. Genet. 1999; 8: 2339-2349Crossref PubMed Scopus (129) Google Scholar, 26Pagani 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 (152) Google Scholar, 27Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar). TDP43 binding to the UG polymorphic repeat reduces the proper recognition of the nearby 3′ splice site and in association with the intronic splicing silencer element in intron 9 mediates exon skipping (27Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar).In this paper, we have analyzed the role of CFTR exon 9 sequences, taking advantage of several natural missense and nonsense mutations and extending them by extensive site-directed mutagenesis. Our results indicate that CFTR exon 9 splicing is extremely sensitive to small variations in its exonic sequence, suggesting that the entire sequence of the exon is important for exon recognition and processing, which occur independently from the maintenance of an open reading frame within the mRNA. We also identify at the 3′ portion of the exon a composite regulatory element with juxtaposed enhancer and silencer properties.EXPERIMENTAL PROCEDURESHybrid Minigene Expression Analysis—The natural and artificial point mutations were introduced in the previously described hCF-(TG)(T) minigenes (26Pagani 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 (152) Google Scholar) between the EcoRI and BamHI sites, which were substituted with the appropriate EcoRI-BamHI cassettes created by PCR-mediated site directed mutagenesis. In hCF, due to the context of the minigene, the natural reading frame of exon 9 (183 bp) was shifted by 1 nucleotide, introducing several stop codons. In the F1 construct, the deletion of a nucleotide in position 16 (T16Δ) and the insertion of a G at position 164 (G144+) produce an open reading frame with both the –1 and +1 exons of the minigene. In the F2 construct, the exon 9 was also placed in an open reading frame by the elimination of a stop codon at the 5′-end of the exon (A20G, TGA->TGG), deleting the nucleotide in position 23 (A23Δ), and inserting a G at position 164 (G164+). The oligonucleotides used for PCR-mediated mutagenesis are available upon request. The exon 9 and flanking intronic sequences of all hybrid minigenes were verified by sequence analysis. Minigene expression analysis was performed on Hep3B cells that were transiently transfected with 3 μg of each minigene plasmids with DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N,-trimethylammonium methyl-sulfate) (Roche Applied Science) and, when indicated, with 500 ng of SF2/ASF coding plasmid. Forty-eight hours post-transfection, RT-PCR was carried out on total RNA as previously described (26Pagani 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 (152) Google Scholar) with the primers 2–3α and B2. Cycloheximide (Gibco) or actinomycin D (Roche Applied Science) were added 48 h post-transfection to final concentrations of 50–2000 and 5 μg/ml, respectively. Cells were collected 72 h post-transfection for cycloheximide treatment or 8, 12, and 24 h post-transfection for actinomycin D. PCRs were optimized to remain in the exponential range of amplification, and products were routinely fractionated in 1.5% (w/v) agarose gel. For quantitation of the PCRs, [α-32P]dCTP was included in the PCR mixture, and the products were loaded on 5% denaturing polyacrylamide, 8 m urea gel, dried, and exposed to a Cyclone Instant Imager phosphorimaging device (PerkinElmer Life Sciences). The counts of each splicing band were corrected by the number of C/G present in the PCR-product sequence.RNA Transcription, UV-cross-linking Assay, and Cross-linking of RNA to Adipic Dehydrazide-Agarose Beads—To generate the WT and mutant RNAs, the different CFTR exon 9 hybrid minigenes were first amplified with the direct primer 5′-tacgtaatacgactcactataggatattaatttcaagatagaaagag-3′, which contains a T7 polymerase sequence and the reverse primer 5′-ctaccttgcctgctccagtg-3′ followed by in vitro T7 RNA polymerase transcription. The amplified region corresponds to the last 64 bp of CFTR exon 9. Transcription of labeled RNAs, nuclear extract preparation, and UV-cross-linking assay were performed as previously described (26Pagani 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 (152) Google Scholar, 27Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar). Cold substrate RNAs for bead immobilization were synthesized by in vitro transcription using T7 RNA polymerase, and cross-linking of RNA to adipic dehydrazide-agarose beads was done essentially as previously described with the addition of heparin to a final concentration of 5 μg/μl (26Pagani 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 (152) Google Scholar, 27Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar). Proteins were separated on a 10% SDS-PAGE gel and visualized by Coomassie Brilliant Blue staining or electroblotted onto a nitrocellulose membrane and probed with a rabbit polyclonal anti-hnRNPH antiserum. Immunoblottings were detected using the ECL chemiluminescence kit (Pierce).RESULTSCFTR Exon Natural Substitutions Can Affect the Splicing Efficiency—To evaluate the contribution of exonic elements in the regulation of CFTR exon 9 alternative splicing, we studied, in the first instance, eight natural point mutations distributed through the entire exon. These substitutions correspond to previously reported seven missense and one nonsense mutations found in classical CF and/or associated with phenotypes of different severity with a tissue specific involvement. Normal or mutated exon 9 sequences were thus inserted in the previously reported hybrid minigene, which has been widely utilized to study alternative splicing regulation of different genes (Fig. 1A) (25Niksic M. Romano M. Buratti E. Pagani F. Baralle F.E. Hum. Mol. Genet. 1999; 8: 2339-2349Crossref PubMed Scopus (129) Google Scholar, 28Caputi M. Casari G. Guenzi S. Tagliabue R. Sidoli A. Melo C.A. Baralle F.E. Nucleic Acids Res. 1994; 22: 1018-1022Crossref PubMed Scopus (137) Google Scholar, 29Cramer P. Pesce C.G. Baralle F.E. Kornblihtt A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11456-11460Crossref PubMed Scopus (266) Google Scholar, 30Pagani F. Buratti E. Stuani C. Bendix R. Dork T. Baralle F.E. Nat. Genet. 2002; 30: 426-429Crossref PubMed Scopus (188) Google Scholar), and in the case of the CFTR exon 9, it has been shown to mimic the endogenous splicing pattern (26Pagani 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 (152) Google Scholar, 27Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar). Since CFTR exon 9 splicing efficiency is modulated by the composition of the polymorphic locus at the 3′-end of intron 8, we first used minigenes with TG11 and T5 repeats. The minigenes were transiently transfected into Hep3B cells, and the splicing pattern was analyzed by RT-PCR amplification. As shown in Fig. 1B, some of the natural substitutions, when compared with wild type hCF, significantly modify the splicing pattern. Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S. The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%). It is interesting to note that three nearby mutations, A146C, C155A, and G157T, at the 3′ portion of the exon have a completely different effect on splicing.To evaluate the effect of exonic substitutions in relation to the strength of the 3′ splice site, we took advantage of the presence of the polymorphic TG and T repeats, which have been shown to modulate the efficiency of the exon recognition. The splicing efficiency of the missense and nonsense variations was analyzed not only in the TG11T5 construct but also in the TG11T9 and TG13T5 minigenes, two variants that cause a respective increase and decrease in the splicing efficiency. Each mutation showed a percentage of exon 9 inclusion to be higher in the presence of the TG11T9, intermediate in the TG11T5, and lower in the TG13T5 context (Fig. 1C). This indicates that both the splicing controlling elements affected by the missense and nonsense mutations along with the 3′ splice site definition independently contribute to the overall efficiency of CFTR exon 9 splicing.Reading Frame Contribution to CFTR Exon 9 Alternative Splicing—Nonsense mutations have been found in some cases to induce abnormal skipping of the exon. We have evaluated the possibility that some of the changes in the splicing pattern induced by the natural substitutions in exon 9, particularly the Q414X, might be related to nonsense mediated altered splicing. The two mechanisms suggested to be involved in the skipping of exons carrying stop codons are a putative nuclear open reading frame scanning process (31Dietz H.C. Am. J. Hum. Genet. 1997; 60: 729-730PubMed Google Scholar) and the contribution of exonic splicing regulatory elements (15Liu H.X. Cartegni L. Zhang M.Q. Krainer A.R. Nat. Genet. 2001; 27: 55-58Crossref PubMed Google Scholar). We evaluated the role of these two mechanisms in the CFTR exon 9 using our minigene system that provides a particular advantage, since alternative splicing produces one mRNA always in an open reading frame (the CFTR exon 9– form) and one mRNA that can contain stop codons according to the reading frame and to the mutation introduced (CFTR exon 9+ form). In the construct containing the normal exon 9 (hCF), the processed CFTR exon 9– mRNA is in frame; however, the CFTR exon 9+ mRNA contains numerous stop codons (Fig. 2A). This is due to the nonnatural context of the minigene, where the presence of globin-fibronectin exons results in a one-nucleotide shift of the reading frame. Transfection of this minigene showed about 65% of exon inclusion. To restore an open reading frame in the minigene that results in the elimination of the multiple stop codons, we prepared the F1 and F2 constructs (Fig. 2A). The open reading frame in both of the mRNAs produced by these minigenes was restored by a base deletion at the 5′-end (F1 contains the T16 deletion, and F2 contains an A23 deletion along with an A20G substitution) and a G insertion at the 3′-end (G164+) (for details, see “Experimental Procedures”). Cotransfection of these two constructs produces about 80% of exon inclusion (Fig. 2D , lanes F1 and F2, respectively). To exclude the possibility that this increase in exon recognition may be due to differential mRNA stability of one of the two alternatively spliced mRNAs (with or without exon 9), the cells were treated with cycloheximide or actinomycin D. Cycloheximide, impairing the efficiency of translation, has been shown to inhibit nonsense-mediated decay (32Noensie E.N. Dietz H.C. Nat. Biotechnol. 2001; 19: 434-439Crossref PubMed Scopus (160) Google Scholar, 33Carter M.S. Doskow J. Morris P. Li S. Nhim R.P. Sandstedt S. Wilkinson M.F. J. Biol. Chem. 1995; 270: 28995-29003Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 34Qian L. Theodor L. Carter M. Vu M.N. Sasaki A.W. Wilkinson M.F. Mol. Cell. Biol. 1993; 13: 1686-1696Crossref PubMed Google Scholar). Analysis of the hCF, F1, and F2 constructs did not show any difference on the relative abundance of the two mRNA variants when treated with the translation inhibitor or actinomycin D (Fig. 2, B and C). This indicates that differential stability of the mRNAs in and out of frame cannot explain the different proportion of plus and minus forms observed.Fig. 2Evaluation of CFTR exon 9 nonsense-associated altered splicing in hybrid minigene experiments. A, schematic representation of the CFTR exon 9 mutants. In the minigene context, hCF contains several stop codons that disappear in F1 and F2, due to the indicated nucleotide deletions and insertions. Alternative splicing of the minigenes produces the CFTR exon 9– form, which is always in an open reading frame, and the CFTR exon 9+ form that may be in an open frame or not according to the mutations introduced in the exon. The bars represent the entire CFTR exon 9 and show the sequence variations and the positions of nonsense codons. The indicated nonsense codons apply only for the exonic sequences in the minigene. The asterisks indicate the two A to T substitutions in position 46 and 49, respectively. B, effect of cycloheximide on the hCF, F1, and F2 constructs. Hep3B cells were transfected with 3 μg of the minigene variants and treated with the indicated concentration of cycloheximide for 24 h as reported under “Experimental Procedures,” followed by radioactive RT-PCR amplification. Each point represents the mean of two independent experiments. C, autoradiography of exon 9+ and exon 9– splicing variants resolved on 6% polyacrylamide gel electrophoresis of actinomycin D-treated cells. Hep3B cells were transfected with hCF, F1, and F2 minigene variants, treated at the indicated time points with actinomycin D (5 μg/ml) as reported under “Experimental Procedures,” followed by radioactive RT-PCR amplification. The numbers below each lane represent the percentage of exon 9 inclusion expressed as the mean of two independent experiments done in duplicate. D, upper panel, autoradiography of exon 9+ and exon 9– splicing variants resolved on 6% polyacrylamide gel electrophoresis. Hep3B cells were transfected with 3 μg of the indicated minigene variants, followed by radioactive RT-PCR amplification. In the lower panel, the RNA splicing variants detected by radioactive PCR were quantitated using a Cyclone. Each bar represents the mean ± S.D. of three independent experiments done in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Thus, the increase in exon recognition of the F1 and F2 constructs compared with hCF may be explained by the interference of the base insertions, deletions, and substitutions with exonic regulatory elements. To fully investigate the putative role of the stop codons in nonsense altered splicing, we introduced in the three hybrid minigenes two A to T substitutions in the central part of the exon (positions 46 and 49 of the exon, respectively) (Fig 2A, Stop constructs). These nucleotide substitutions, according to the different reading frames of the hCF, F1, and F2 minigenes, do or do not generate stop codons. In the F1 and F2 contexts, these nucleotide changes generate two unique stop codons. The transfection experiments showed no changes in the splicing patterns with about 80% of exon inclusion (Fig. 2D , F1-Stop and F2-Stop). On the other hand, in the hCF minigene, the two A to T substitutions eliminated one stop codon, but again the splicing pattern was not affected, remaining at the initial 65% for this construct. This indicates that the two A to T substitutions, even if they produce nonsense codons, do not induce altered proportions of plus and minus forms, since they are not affecting splicing regulatory elements. We then tested, in the three different contexts, two of the natural substitutions with a splicing-inhibitory effect, the C31T and the C155A. In the hCF context, the C31T and the C155A do not introduce a new stop codon but reduce the 65% hCF exon inclusion to 48 and 16%, respectively (Fig. 2D, compare hCF with hCF-C31T and hCF-C155A). On the other hand, in the F1 and F2 minigenes, C31T creates a stop codon (Q414X), whereas C155A does not. The results shown in Fig. 2D indicate that these two mutations maintain a similar splicing-inhibitory effect both in F1 and F2 contexts. In fact, the 80% of exon inclusion in F1 and F2 is reduced in F1-C31T and F2-C31T to about 65%, and in F1-C155A and F2-C155A it is reduced to about 35% (Fig. 2D). These results indicate that the mutations have a negative effect on the exon recognition independently from the nonsense codon specification. In addition, we introduced on the F2 context two different stop codons in the same 5′-end position, with the creation of the F2-TAA and F2-TGA constructs. In one case, the stop codon (TAA) did not change the splicing pattern, whereas a 70% exon inclusion was observed for the TGA codon (Fig. 2D).Altogether, these results indicate that substitutions introduced in the exon may affect the exon recognition, but this is completely independent from the fact that the substitutions do or do not create a stop codon. In conclusion, these data exclude the presence of a nuclear reading frame mechanism regulating CFTR exon 9 splicing and suggest that exonic mutations, including nonsense substitutions, cause changes in the splicing efficiency by affecting exonic splicing-controlling elements.Identification of Regulatory Elements of Splicing in CFTR Exon 9 —Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides. This suggests that this short sequence at the 3′ end of exon 9 may contain both enhancer and silencer functions, whose fine tuning on splicing could be missed if analyzed using multiple base deletions. To better characterize the mechanism by which some of the CFTR exon 9 mutations alter splicing, we performed a detailed investigation by site-directed mutagenesis of this short region (positions 144–157). This analysis was further extended to other point mutations distributed in the entire length of the exon (Fig. 3).Fig. 3Nucleotide sequence of the entire CFTR exon 9 showing the location of missense, nonsen