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.

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-activated Cl Ϫ channels (CaCCs) 2 play a major physiological role in various types of cells and tissues (1-4). In epithelial cells, opening of CaCCs in the apical membrane generates a flux of Cl Ϫ that drives transepithelial water transport. The resulting CaCC-dependent electrolyte/fluid secretion is one of the mechanisms responsible for exocrine secretion in many types of glands and for hydration of the airway surface (2). In particular, the activity of CaCC and of the cystic fibrosis transmembrane conductance regulator chloride channel controls the thickness of the periciliary fluid that is important for optimal mucociliary clearance (5, 6). Deficit in cystic fibrosis transmembrane conductance regulator activity, which occurs in cystic fibrosis, favors bacterial colonization of the airways. Under such conditions, activity of CaCCs may be important in compensating, at least partially, the defect in Cl Ϫ transport. In smooth muscle cells, activation of CaCCs is important in the process of contraction (1,7,8). CaCCs are also present in olfactory receptors, in dorsal root ganglion neurones, and in oocytes (3, 4, 9, 10). The biophysical properties of CaCCs are not homogeneous among different cells and tissues. In many cases, CaCCs are activated by Ca 2ϩ in a wide range of concentrations between 0.06 and 1 M (11-14) and are also voltage-dependent, with membrane depolarization increasing the activity (7, 10 -16). However, CaCCs that need much higher Ca 2ϩ concentration (9, 17, 18) and that are devoid of voltage dependence are also known (9,17,19). Moreover, the mechanism of regulation by Ca 2ϩ is unclear. In some studies, Ca 2ϩ seems to activate the channels through calmodulin (20). In others, activation requires the intervention of a Ca 2ϩ /calmodulin-dependent kinase (19,21). Finally, there are cell types where phosphorylation has actually an inhibitory effect on CaCCs (7,8,14). The differences may be due to heterogeneity of the proteins that actually constitute the CaCCs.Until recently, the molecular identity of CaCCs was a controversial issue, with ClCA proteins, ClC3, and bestrophins being postulated as possible candidates (1,22). Three recent studies, including one from our laboratory, have identified the TMEM16A protein (also known as ANO-1) as a probable . TMEM16A is a membrane protein with eight putative transmembrane segments belonging to a family including other nine members (TMEM16B-K). In our previous study (23), we identified several TMEM16A transcripts probably generated by selection of alternative splice sites. The alternative sequences coded for protein segments that we named a (116 residues), b (22 residues), c (4 residues), and d (26 residues). The former two segments are localized in the N terminus, whereas the latter two segments are localized in the first intracellular loop.A TMEM16A mouse knock-out model has been also generated. The phenotype of these animals is severe and characterized by altered formation of tracheal cartilage rings (26). This alteration, causing airway collapse, may be resp...

Disease-causing splicing mutations described in the literature primarily produce changes in splice sites and, to a lesser extent, variations in exon-regulatory sequences such as the enhancer elements. The gene ATM is mutated in individuals with ataxia-telangiectasia; we have identified the aberrant inclusion of a cryptic exon of 65 bp in one affected individual with a deletion of four nucleotides (GTAA) in intron 20. The deletion is located 12 bp downstream and 53 bp upstream from the 5' and 3' ends of the cryptic exon, respectively. Through analysis of the splicing defect using a hybrid minigene system, we identified a new intron-splicing processing element (ISPE) complementary to U1 snRNA, the RNA component of the U1 small nuclear ribonucleoprotein (snRNP). This element mediates accurate intron processing and interacts specifically with U1 snRNP particles. The 4-nt deletion completely abolished this interaction, causing activation of the cryptic exon. On the basis of this analysis, we describe a new type of U1 snRNP binding site in an intron that is essential for accurate intron removal. Deletion of this sequence is directly involved in the splicing processing defect.

It is well established that exonic sequences contain regulatory elements of splicing that overlap with coding capacity. However, the conflict between ensuring splicing efficiency and preserving the coding capacity for an optimal protein during evolution has not been specifically analyzed. In fact, studies on genomic variability in fields as diverse as clinical genetics and molecular evolution mainly focus on the effect of mutations on protein function. Synonymous variations, in particular, are assumed to be functionally neutral both in clinical diagnosis and when measuring evolutionary distances between species. Using the cystic fibrosis transmembrane conductance regulator (CFTR) exon 12 splicing as a model, we have established that about one quarter of synonymous variations result in exon skipping and, hence, in an inactive CFTR protein. Furthermore, comparative splicing evaluation of mammalian sequence divergences showed that artificial combinations of CFTR exon 12 synonymous and nonsynonymous substitutions are incompatible with normal RNA processing. In particular, the combination of the mouse synonymous with the human missense variations causes exon skipping. It follows that there are two sequential levels at which evolutionary selection of genomic variants take place: splicing control and protein function optimization.composite exonic regulatory elements of splicing ͉ exonic splicing regulatory sequences ͉ molecular evolution ͉ synonymous variations S ince the first original observations that nucleotide changes in the coding regions can affect normal and alternative cellular pre-mRNA processing (1, 2), extensive evidence has accumulated that exonic variants may affect pre-mRNA splicing (3-6). Mutations within exons are responsible of aberrant splicing profiles of pre-mRNA in several human disease genes, including ataxia telangiectasia mutated (ATM) (7), SMN2 (8-10), BRCA1 (11), neurofibromin 1 (NF-1) (12), cystic fibrosis transmembrane regulator (CFTR) (13-15) genes and in some viral systems such as HIV-1 (16). Nonsense, missense, and even synonymous mutations can induce aberrant skipping of the mutant exon producing nonfunctional proteins. Because direct analysis of mRNA is not routinely performed in the diagnostic field, it is possible that a high amount of exonic mutations may unexpectedly affect splicing, as reported in ATM (7), CFTR (13-15), and NF-1 (12) genes.In the pre-mRNA of higher eukaryotes, the correct identification of exonic sequences from the larger introns requires conserved discrete sequence elements located at the 5Ј and 3Ј splice sites and the branch point. In addition, exonic splicing elements that interact with several classes of positive and negative trans-acting splicing regulatory factors contribute significantly to constitutive and alternative splicing regulation (3, 17). The sequence composition of exonic splicing regulatory sequences is highly degenerated (3,(18)(19)(20)(21), and their effect on splicing may be significantly affected by the context (13,14) and͞or depend on the fo...