Figure 3

Mutations at Aldh1A2 exon 4 splicing enhancer alter splicing efficiency. A) c.G451T and c.A453G of the Aldh1a2 exon 4 interrupt a putative binding site for the splicing factor SF2/ASF. B) In pSPL3 splicing assay a long HIV tat intron is flanked by two exons containing weak splicing signals. Fragments inserted within the pSPL3 intron are spliced according to the potency of their signals. C) If splicing signals in wild type ALDH1A2 exon 4 are strong, the assay will produce a 250 bp PCR fragment containing Aldh1a2 exon 4 flanked by HIV exons 1 and 2. If exon 4 mutants reduce the efficiency of splicing signals a PCR fragment containing HIV exons 1 and 2 + ALDH1A2 exon 4 will be produced (380 bp). D) pSPL3 assays with HEK 293 cells indicate that the wild-type exon 4 contains weak splicing signals, as indicated by production of PCR fragments without exon 4 sequences. The c.G451T transversion strengthens exon 4 splicing signals, as indicated by the disappearance of the smaller (250 bp) fragment. First lane, 100 bp ladder; second lane, pSPL3 vector; third lane pSPL3 vector + fragment of the human ALDH1A2 gene containing the wild type exon 4 flanked by about 450 bp of intronic sequences; forth lane, pSPL3 vector + fragment of the human ALDH1A2 gene containing the c.G451T transversion, flanked by about 450 bp of intronic sequences. E) The c.A453G transition is associated with a small, non-significant decrease in splicing strength (4%). First lane, pSPL3 vector; second lane, pSPL3 vector + fragment of the human ALDH1A2 gene containing the wild type exon 4 flanked by about 450 bp of intronic sequences; third lane, pSPL3 vector + fragment of the human ALDH1A2 gene containing the c.A453G, flanked by about 450 bp of intronic sequences.