RNA splicing mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein have been described to lead to dysfunction of several organs such as lung, sweat glands, genital tract, intestine and pancreas, producing the complex CF symptoms (Welsh et al., 1995). CFTR mutations can also be associated with a variety of isolated clinical signs such as congenital bilateral absence of vas deferens (CBAVD) (Chillon et al., 1995; Dörk et al., 1997), nasal polyposis (Irving et al., 1997), bronchiectasis (Pignatti et al., 1995; Girodon et al., 1997), bronchopulmonary allergic aspergillosis (Cockrill and Hales, 1999) or idiopathic pancreatitis (Cohn et al., 1998; Sharer et al., 1998). In particular, the occurrence of CBAVD has been associated with production of an inactive CFTR protein following the loss of exon 9 from the coding mRNA through a process of aberrant alternative splicing (Delaney et al., 1993; Strong et al., 1993).
Several genetic studies have thus been aimed at identifying the cis-acting elements on the human CFTR gene in the vicinity of exon 9 that might explain this unusual splicing process. The elements identified so far (Figure 1A) include a (TG)m(T)n polymorphic element, the recently identified intronic splicing silencer (ISS) in IVS9, and two exon 9 enhancer and silencer elements (Pagani et al., 2000). Initially, variability in a (T)n polymorphic locus located within the 3′ splice site of IVS8 was the first element to be associated with a variable efficiency of exon 9 splicing (Chu et al., 1991). The high proportion of a T5 allele in patients affected by male infertility (caused by obstructive azoospermia, e.g. CBAVD) represents one of the better characterized correlations between the occurrence of a particular polymorphism and the associated clinical signs (Chu et al., 1993; Chillon et al., 1995; Mak et al., 1997; Rave-Harel et al., 1997; Teng et al., 1997; Cuppens et al., 1998; Larriba et al., 1998). Nonetheless, the T5 allele effect has partial penetrance, making it possible to find healthy homozygous carriers. Recently, a second polymorphic locus based on (TG)m repeats (ranging from 9 to 13 repeats in humans) localized immediately upstream of the (T)n tract was found to influence the efficiency of exon 9 splicing (Cuppens et al., 1998). In particular, T5 CFTR genes derived from CBAVD patients carried a high number of TG repeats, whilst T5 CFTR genes derived from healthy fathers carried a low number of TG repeats (Cuppens et al., 1998), suggesting that this element may play a role in the partial penetrance of the T5 allele. In previous studies we have specifically analyzed, using a minigene system, the effect of these two cis-acting elements on exon 9 splicing, and our results confirmed that the (TG)m and (T)n repeats work in concert with each other (Niksic et al., 1999). Moreover, the identification and characterization (in this work) of a CF patient carrying the TG13T3 genotype indicates that the (TG)m(T)n variability is not only associated with monosymptomatic forms of CF but that its extreme variant may also be associated with pancreatic-sufficient CF. Therefore, the identification of the cellular factors eventually binding to these elements represents a key step in understanding the complex regulation of exon 9 splicing. In this study, we identify HIV-1 TAR DNA binding protein (TDP-43) (Ou et al., 1995) as a novel factor binding to the TG element in CFTR exon 9 pre-mRNA and capable of modulating CFTR exon 9 alternative splicing. Most importantly, antisense inhibition of endogenous TDP-43 results in an upregulation of exon 9 inclusion, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients.