Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element

In monosymptomatic forms of cystic fibrosis such as congenital bilateral ab sence of vas deferens, variations in the TG(m) and T-n polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (C FTR) gene are associated with the alternative splicing of exon 9, which res ults in a nonfunctional CFTR protein. Using a minigene model system, we hav e previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-r ich proteins and the heterogeneous nuclear ribonucleoprotein Al induced exo n skipping in the human gene but not in its mouse counterpart. The effect o f these proteins on exon 9 exclusion was strictly dependent on the composit ion of the TG(m) and T-n polymorphic repeats. The comparative and functiona l analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 sp licing inhibition in association with exonic regulatory elements. This regi on, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conser ved CFTR exon 9 alternative splicing is modulated by the TG(m) and T-n poly morphism at the 3' splice region, enhancer and silencer exonic elements, an d the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibros is.