(A) Shows the sequence of the entire IVS9 (h3′int) sequence from the last nucleotides of exon 9 (in capital letters) to the NdeI site that was cloned in the TG11T5 original minigene. Also shown in this diagram are the positions of the two mutated nucleotides that create a unique PstI site. In the ISS region, the sequences targeted by each of the four antisense oligos (AS1–AS4) are underlined. (B) and (C) shows the immunoprecipitation profiles of the h3′int sequence using mAb 96 (B) and mAb 1H4 (C) monoclonal antibodies in the presence of each of the four antisense oligos. The migration of SF2/ASF and of SRp40 are marked with an arrow.

(A) Upper panel shows a comparison between the AS1 + AS2 sequence and the heterologous polypurinic sequences from the EDA ESE and the Apo AII ISE elements. The column on the right shows the SR protein-binding specificity of all these sequences as previously determined by immunoprecipitation analysis. The EDA ESE and Apo ISE sequences were then inserted in the pES plasmid (as shown in the lower panel), thus creating constructs pTB EDAPK and pTB ApoISEPK, respectively. The levels of CFTR exon 9 inclusion displayed by these three plasmids in Hep3B cells and compared with that of the pES and pTB AS1 + AS2PK constructs is reported in (B). A quantification of three independent experiments is reported in (C). (D) Shows the response of the TG11T5, pTB AS1 + AS2PK, pTB EDAPK and pES plasmids to SF2/ASF overexpression, and of the response of the pTB AS1 + AS2PK construct to SC35 overexpression, following transfection in Hep3B cells.

(A) Shows the sequence of the YB-1-binding motif inserted in the PstI/KpnI restriction sites of the pES plasmid (pTB YB1PK). (B) Left panel shows a comparison of the levels of CFTR exon 9 inclusion in the TG11T5, pES and pTB YB1PK plasmids following transfection in Hep3B cells. The position of the transcripts including exon 9 (ex9+) and lacking exon 9 (ex9−) are marked on the right. The results of three independent experiments as quantified by radioactive RT–PCR are reported in (B), right panel. The effect of cloning the YB-1 sequence only in the PstI site of the pES plasmid (pTB YB-1P) as opposed to the PstI/KpnI sites (pTB YB-1PK) is shown in (C). The schematic diagram on the left shows the composition of the unique splice product observed in lane 1 whilst the position of the transcripts including exon 9 (ex9+) and lacking exon 9 (ex9−) are marked on the right.

(A) Shows a schematic diagram of the splicing controlling regions of CFTR exon 9, both within the exon (CERES element) and in the flanking IVS8 and IVS9 sequences: TG(m)T(n), PCE and ISS. h3′int defines the IVS9 region that includes both the PCE and ISS controlling elements. (B) Shows the trans-acting factors identified up to now that bind to these elements. (C) Shows an immunoprecipitation analysis of the h3′int region and of the fibronectin EDA ESE element both in its wild-type (hTot) and mutated form (h▵2e). The left, central and right panels show the immunoprecipitation profiles obtained from each RNA using mAb 96 (specific against SF2/ASF), mAb 1H4 (specific against the phosphorylated RS domain) and an anti-SC35 antibody.

(A) Upper panel shows a schematic representation of the pES hybrid minigene carrying the CFTR exon 9 along with intronic flanking sequence. Black, shaded and white boxes represent alpha globin, fibronectin and CFTR exons, respectively. The arrows indicate the position of the primers used to amplify the processed mRNA species. The dotted lines represent the expected splicing pattern that determines exon 9 inclusion/exclusion. Lower panel shows a close-up view of the nucleotide composition of the IVS9 sequence in the PstI/KpnI/NdeI cloning region. The black box represents the exact position in which the AS1 + AS2 (pTB AS1 + AS2PK) and AS3 + AS4 (pTB AS3 + AS4PK) sequences were inserted. (B) Shows a comparison of the RT–PCR splicing profiles of these two constructs together with the pES and TG11T5 reference minigenes following transfection in Hep3B cells. The position of the transcripts including exon 9 (ex9+) and lacking exon 9 (ex9−) are marked on the right. The results of three independent experiments were quantified using radioactive PCR and are reported in (C) with SD values. (D) Shows a set of analogous transfections in Hep3B cells using a set of plasmids in which each individual AS1, AS2, AS3 and AS4 sequences was inserted in the PstI/KpnI sites of the pES plasmid (pTB-AS1PK to pTB-AS4PK). A quantification of the effect of these sequences on CFTR exon 9 inclusion is reported in (E).

(A) Shows a comparison of the different splice site composition when the AS1 + AS2, EDA and ApoISE sequences are cloned in the PstI/KpnI sites (upper panel, PK plasmid series) as opposed to the PstI site alone (lower panel, P plasmid series). The differing splice site composition in the region surrounding the point of insertion is indicated by arrows, with the conserved 3′ss ‘ag’ and 5′ss ‘gt’ nucleotides highlighted in bold. (B) Shows a comparison of the splicing profiles between P and PK plasmids carrying the AS1 + AS2, EDA and ApoISE sequence following transfection in Hep3B cells. The asterisk shows the extra band that is observed in the plasmids carrying the heterologous donor site downstream of the inserted sequence (pTB AS1 + AS2P, pTB EDAP and pTB ApoISEP). The lower panel shows a schematic diagram of the new splicing event indicated by the asterisk whilst the position of the transcripts including exon 9 (ex9+) and lacking exon 9 (ex9−) are marked on the right. (C) Left panel shows a transfection analysis of a TG11T5 mutant (IVS9del3′ss) carrying a ‘ag’ to ‘aa’ mutation in the cryptic 3′ss sequence. The CFTR exon 9 inclusion levels are comparable to those detected for the TG11T5 wild-type plasmid (C, right panel).

This figure shows a schematic diagram of different splicing systems in which SR proteins have been observed to behave as inhibitors of exon inclusion. (A) Shows the CE9 element characterized in the hnRNP A1 gene, (B) to the ISE element observed downstream of b-tropomyosin exon 6A, (C) to the 3RE element present in Adenovirus and (D) to the NRS element present in Rous Sarcoma Virus. Finally, a working model of the way SR proteins may inhibit CFTR exon 9 recognition is reported in (E).