Exon Specific U1s recover CFTR protein levels in exon 13 and 16
mutant minigenes
To evaluate if the splicing correction mediated by Exon Specific U1
rescues the CFTR protein, we focussed on exon 13 and 16. The WT and
mutant CFTR exons were inserted into cDNA splicing competent minigenes
that code for the entire CFTR protein in a manner that the exon
inclusion mRNA variants will result in the production of a normal CFTR
proteins. The minigenes were transfected along with each active ExSpeU1s
and CFTR protein analysed by Western blotting. As expected from the
severity of the splicing defect (Fig.1C), the two exon 13 mutants,
1898+3A>G and 1863C>T (Y577Y) did not show any
detectable CFTR mature protein in Western blotting (Fig.2A, lanes 4 and
5). Co-transfection of U1ex13-11 rescued both the exon skipping defects
(Fig.1C) with production of the full-length fully-glycosylated 160KD
form of CFTR protein (Fig. 2A, lanes 6 and 7). Control full-length CFTR
cDNA and a minigene with the WT exon 13 showed the mature CFTR form
(Fig.2A lanes 1 and 3, respectively) whereas a minigene without exon 13
did not show any band (Fig. 2A, lane 2). Consistent with a residual
splicing activity (Fig.1), the mutation 2789+5G>A mutation
in exon 16 showed a reduced amount of the CFTR mature form, compared to
the WT minigene (Fig. 2B, compare lanes 1 and 2). Treatment with the
three active ExSpeU1s, U1ex16(-3), U1ex16-7 and U1ex16-12) showed a
two-fold increase in the amount of mature CFTR protein of 160kDa (Fig.
2B, lanes 3, 4 and 5).