Strict requirement of RCAG/G in Fas intron 5 3′ splice site. (A) Fas minigenes encompassing exon 5 to exon 7 carrying the wild-type sequence or C-to-G mutation at position −3 of intron 5 (referred to as AG because it generates a duplicated AG dinucleotide at the 3′ splice site) found in an ALPS patient were transfected in HeLa cells. RNA was extracted, and an alternative splicing pattern was monitored by RT followed by semi-quantitative RT-PCR. The alternatively spliced products were resolved by agarose gel electrophoresis. The positions of size markers and predicted alternatively spliced products are indicated. Average percentage of inclusion (% incl.) for a minimum of three independent experiments and SD values are indicated. (B) Splicing pattern of wild-type and AG minigenes containing single introns. Analyses were as in A using primers appropriate to detect spliced and intron retention products. The vertical line in intron 6 represents a deletion that facilitates the detection of unspliced RNA but that does not affect Fas splicing or regulation (Izquierdo et al. 2005). (C) Sequence of Fas intron 5 3′ splice site region. Numbers indicate the distance from exon 6, which is represented by a black box. (D) Mutational analysis of intron 5 3′ splice site region. The indicated minigene mutants were transfected in HeLa cells and the alternative splicing pattern monitored as in A. Lanes 1 and 2 correspond to the wild-type and AG minigenes, respectively. (E) Mutational analysis of exon 6 positions +1 and +2. (F) Reconstructing an ACAGG sequence (underlined) suppresses the effects of the AG mutant and activates the mutant AG. The indicated 3′ splice site region mutants were analyzed as in D. (/) Original intron/exon boundary; (AG, bold) AG generated by the ALPS mutation; (gray boxes) acceptor site used when exon 6 is included.
