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.