Alternative splicing of human cystic ®brosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both¯anking introns (IVS8 and IVS9). Several studies have identi®ed in the IVS8 intron 3¢ splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identi®ed that recognize this element. We have identi®ed TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding speci®c-ally to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this ®nding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DF508)/ TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence 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 (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have 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-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG m and T n polymorphic repeats. The comparative and functional 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 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG m and T n polymorphism at the 3 splice region, enhancer and silencer exonic elements, and 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 fibrosis. Cystic fibrosis (CF),1 the most common life-shortening autosomal recessive disorder in Caucasians, is caused by mutations in the CF transmembrane regulator (CFTR) gene and is characterized by pathological features of variable severity at the level of lungs, pancreas, sweat glands, testis, ovaries, and intestine (1). Monosymptomatic forms of the disease such as congenital bilateral absence of vas deference (CBAVD), pancreatitis, nasal polyposis, disseminated bronchiectasies, and broncopulmonary allergic aspergillosis frequently present a peculiar allele at the polymorphic CFTR intron 8-exon 9 junction (2-8). At this locus a variable number of dinucleotide TG repeats (from 9 to 13) followed by a T repeat (T5, T7, or T9) can be found in the normal population, and it has been suggested that the T5 allele is a disease mutation with incomplete penetrance that could be modulated by the simultaneous presence of other mutations and/or polymorphisms (3). The pathologic effect of the T5 allele has been associated to the alternative splicing of the CFTR exon 9, which is extremely variable in humans among different individuals (9). Interestingly, exon 9 skipping is absent in mouse, and it has been reported not to be evolutionary conserved (10). This exon encodes part of the functionally important first nucleotide-binding domain, and its skipping produces a nonfunctional CFTR protein (10, 11). In CBAVD patients ...

Fas ligand (FasL), a potent mediator of apoptosis expressed by CTL and NK cells, is sorted into the inner vesicles of secretory lysosomes for release via exosome-like vesicles. Previous studies identified a proline-rich domain in the cytoplasmic tail required for sorting FasL to secretory lysosomes, but the mechanisms by which this occurs have not been identified. Here we demonstrate that the PRD of FasL binds Fgr, Fyn and Lyn tyrosine kinases, leading to phosphorylation of FasL. Loss of phosphorylation reduces internalisation of FasL into multivesicular bodies. FasL is also directly mono-ubiquitylated at lysines flanking the PRD and mutation of these lysines reduces MVB localisation of FasL. Phosphorylation is not required for ubiquitylation because FasL lacking all tyrosines undergoes mono-ubiquitylation. These studies show that phosphorylation and ubiquitin signals regulate the sorting of FasL to secretory lysosomes by controlling entry into multivesicular bodies.

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041-21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774 -1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tractbinding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1. Cystic fibrosis (CF)1 is the most common autosomal recessive disorder in Caucasians, and it is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1). CFTR mutations can also be associated with nonclassical forms of CF in which the disease shows a tissuespecific variability such as congenital bilateral absence of vas deference and idiopathic pancreatitis (2). In some cases, the phenotypic variability has been associated with a variable proportion of aberrant CFTR exon 9 skipping, which produces a nonfunctional protein (3-7) and can be modulated by splicing factors. Extensive studies on CFTR exon 9 alternative splicing have provided a paradigmatic example of the complexity of its regulation and, in this case, the possibility of aberrant exon skipping that leads to pathologi...

Using hybrid minigene experiments, we have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, cystic fibrosis transmembrane regulator (CFTR) exon 9 and fibronectin extra domain-A (EDB). A specific alternative splicing pattern corresponded to each analyzed promoter. Promoter-dependent sensitivity to cotransfected regulatory splicing factor SF2/ASF was observed only for the CFTR exon 9, whereas that of the EDB was refractory to promoter-mediated regulation. Deletion in the CFTR minigene of the downstream intronic splicing silencer element binding SF2/ASF abolished the specific promotermediated response to this splicing factor. A systematic analysis of the regulatory cis-acting elements showed that in the presence of suboptimal splice sites or by deletion of exonic enhancer elements the promoter-dependent sensitivity to splicing factor-mediated inhibition was lost. However, the basal regulatory effect of each promoter was preserved. The complex relationships between the promoter-dependent sensitivity to SF2 modulated by the exon 9 definition suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation.In most eukaryotic cells, transcription and pre-mRNA processing (capping, splicing, and cleavage/polyadenylation) are coordinately regulated within the nucleus both in a temporal and spatial fashion (for review see Refs. 1-3). The phosphorylated C-terminal domain (CTD) 1 of RNA polymerase II (pol II) provides key molecular contacts with these mRNA processing reactions throughout transcriptional elongation and termination. For example, transcripts originating from polymerases without a CTD (T7 polymerase and pol III) cannot be spliced or polyadenylated and, indeed, expression of a form of pol II lacking the CTD does not abrogate transcriptional activity but actually depresses pre-mRNA processing (4). The CTD can physically interact with several pre-mRNA processing factors (4 -8), including SR proteins (9), and with transcriptional factors that may have a dual role in transcription and splicing regulation.The connection between transcription and splicing might have important functional implications in vivo, because the promoter-dependent recruitment of regulatory splicing factor and/or changes in RNA pol II elongation and/or its phosphorylation state may influence the subsequent splice site selection (10). EDA transcript can be modified by changes in the promoter region of the gene, using a transient expression system combined with a promoter swapping processing of the alternative splicing fibronectin (11). It has been clearly shown that this effect is not because of the different mRNA levels produced by each promoter but is related to qualitative properties of the promoters. In addition, the sensitivity to overexpressed SR proteins, which induce EDA exon inclusion, depends on the promoter, thus suggesting that the promoter can modulate regulatory splicing factor action through the partic...