CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3 (original) (raw)
Related papers
Journal of Biological Chemistry, 2000
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.
Journal of Biological Chemistry, 2003
Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3 portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms.
Splicing factors induce CFTR exon 9 skipping through a non-evolutionary conserved intronic element
2000
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.
Functional Analysis of cis-Acting Elements Regulating the Alternative Splicing of Human CFTR Exon 9
Human Molecular Genetics, 1999
The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3′ ′ ′ ′ splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG) n repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3′ ′ ′ ′ splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3′ ′ ′ ′ splice site sequences, the extent of exon 9 skipping in CFTR mRNA.
Journal of Biological Chemistry, 2004
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 () 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 3splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1.
Nuclear factor TDP43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping
Embo Journal, 2001
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®cally 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.
Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts
Human Genetics, 1992
Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts isolated from both expressing and "non-expressing" cell types of normal individuals exhibit differntial splicing to a variable extent in a region encoding the putative nucleotide binding fold of the CFTR polypeptide. Sequence analysis of the aberrant fragments obtained after cDNA polymerase chain reaction amplification confirmed the in-frame joining of exons 11 and 13. The proportion of alternative splicing is reproducible and constant in a given individual. The omission of exon 12 in a significant proportion of transcripts supports the hypothesis that a minimal amount of correctly expressed CFTR is sufficient for the maintenance of a clinically normal phenotype.
FEBS Letters, 1993
The cystic fibrosis transmembrane conductance regulator (CFTR) has been extensively characterized as the carrier of the basic defect in cystic fibrosis. CFTR is part of a growing family of proteins encoded by a single gene, the variant isoforms of which are generated by alternative splicing or RNA editing. We have analyzed the CFTR mRNA in the region of exons 10-l 1 in T&l cells and detected an alternatively spliced exon (lob) accounting for about 5% of the CFTR mRNA. The exon lob found in both the human and mice genomes, introduces an in-frame stop codon. The resulting mRNA is translated into a truncated CFTR protein, identified in T84 cells by immunoprecipitation with the CFTR-specific monoclonal antibody MATG 1061. The insertion of a differentially spliced exon carrying an in-frame stop codon is a novel cellular mechanism for the production of a protein sharing common sequences with another, but having different properties and functions.
hnRNP A1 induces aberrant CFTR exon 9 splicingviaa newly discovered Exonic Splicing Silencer element
RNA-protein interactions play a key role in the aberrant splicing of CFTR exon 9. Exon 9 skipping leads to the production of a non-functional chloride channel associated with severe forms of cystic fibrosis. The missplicing depends primarily on variations in the polymorphic (TG)mTnlocus upstream of exon 9. At the pre-mRNA level, it generates an extended UG-rich binding site for TDP-43, associated with hnRNP A1 recruitment, and prevention of exon 9 3’ splicing site (3’ss) recognition. While TDP-43 is the dominant inhibitor of exon 9 inclusion, the role of hnRNP A1, a protein with two RNA recognition motifs (RRM1 and RRM2) and a glycine-rich domain, remained unclear. In this work, we have studied the interaction between hnRNP A1 and the CFTR pre-mRNA using NMR spectroscopy and Isothermal Thermal Calorimetry (ITC). The affinities are submicromolar and ITC data suggest that the separate RRMs as well as tandem RRMs form 1:1 complexes. NMR titrations reveal that hnRNP A1 interacts with mo...
Journal of Biological Chemistry, 2001
Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.