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).