CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain.