Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conduct ance regulator (CFTR) mRNA is associated with monosymptomatic forms of cyst ic fibrosis, Exon 9 alternative splicing is modulated by a polymorphic poly thymidine tract within its 3' splice site, We have generated a minigene car rying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splic ing, Transfections into human cell lines showed that T5, but not T9 or T7 a lleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of th e T tract, and constituted by (TG)(n) repeats, can further modulate exon 9 skipping but only when activated by the T5 allele, Then, we extended our st udies to the mouse CFTR exon 9 which does not show alternative splicing, Co mparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site, We have tested a series of constructs where t he whole human exon 9 with its flanking intronic sequences was replaced par tially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/ U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA.