Abstract
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.
Highlights
Skipping of constitutive exons may occur for missense and silent mutations
Our results indicate that CFTR exon 9 splicing is extremely sensitive to small variations in its exonic sequence, suggesting that the entire sequence of the exon is important for exon recognition and processing, which occur independently from the maintenance of an open reading frame within the mRNA
CFTR Exon Natural Substitutions Can Affect the Splicing Efficiency—To evaluate the contribution of exonic elements in the regulation of CFTR exon 9 alternative splicing, we studied, in the first instance, eight natural point mutations distributed through the entire exon
Summary
Hybrid Minigene Expression Analysis—The natural and artificial point mutations were introduced in the previously described hCF(TG)(T) minigenes [26] between the EcoRI and BamHI sites, which were substituted with the appropriate EcoRI-BamHI cassettes created by PCR-mediated site directed mutagenesis. In the F1 construct, the deletion of a nucleotide in position 16 (T16⌬) and the insertion of a G at position 164 (G144ϩ) produce an open reading frame with both the Ϫ1 and ϩ1 exons of the minigene. Cold substrate RNAs for bead immobilization were synthesized by in vitro transcription using T7 RNA polymerase, and cross-linking of RNA to adipic dehydrazide-agarose beads was done essentially as previously described with the addition of heparin to a final concentration of 5 g/l [26, 27].
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