Figure legends
Figure 1: Exon Specific U1s rescue splicing defects that cause skipping of CFTR exons. Panels A), B), C), D) and E) show minigene splicing assay for CFTR exons 5, 9, 13, 16 and 18, respectively. The indicated WT and mutant minigene variants were transfected in HeLa cells alone or co-transfected with the indicated ExSpeU1s. In each panel, the upper part shows a schematic representation of CFTR exons, mutants and ExSpeU1s; the intermediate figure shows the agarose gel and the lower panel the percentage of exon inclusion. Identity of the exon inclusion and skipping band are indicated. In B), the middle band corresponds to the activation of a previously described cryptic splice site (Buratti et al., 2001).
Figure 2: Exon Specific U1s rescue CFTR protein levels in exon 13 and exon 16 splicing mutations. For each panel the upper part shows schematic representation of CFTR exons along with mutants and position of the ExSpeU1s, the intermediate figure shows the Western Blot analysis and the lower panel the percentage of CFTR protein normalized over Tubulin. Data are expressed as means ± SD of three independent experiments done in duplicate. A) Western Blot analysis of exon 13 with total CFTR cDNA, CFTR cDNA without Exon13, CFTR cDNA flanked by partial sequence of intron 12 and 13, 1863C>T, 1898+3A>G mutants and ExSpeU1. B) Western Blot analysis of exon 16 with WT, 2789+5G>A mutant and ExSpeU1s.
Figure 3: The Intronic Splicing Silencer (ISS) in CFTR intron 13 overlaps with the U1ex13-11 binding site. A) Effect of consecutive intron 13 deletions on CFTR exon 13 splicing in minigene assay in WT, 1863C>T and 1898+3A>G variants. The upper panel indicates the exon13-intron 13 region along with the position of intronic deletions and mutants. Position +1 is the first base of the intron. The intermediate panel is a representative agarose gel of the splicing assay and the lower graph shows of the percentage of exon inclusion expressed as the means ± SD of three independent experiments done in duplicate. B) Predicted RNA secondary structure of CFTR exon 13-intron 13 region (RNA m-fold analysis). The ISS is indicated by a black line. Exonic sequences are in the grey box and intronic sequences are in lowercase. Position +1 is the first base of the intron. C) Fine mapping of the ISS in WT context. The upper panel shows the sequence of the exon13-intron 13 junction along with the position of the ISS point mutations from position 12 to 23. Position +1 is the first base of the intron. The intermediate panel is a representative agarose gel of the splicing assay of the indicated single or double mutant minigenes. The lower graph shows the percentage of exon inclusion expressed as the means ± SD of three independent experiments done in duplicate. D) The relationship between percentage of exon 13 inclusion (y axis) and changes in estimated ΔG (x axis) fitted in sigmoidal function.