Figure 5. Exontrap assays demonstrate the functional effect of rs1131454 on OAS1 exon 3 splicing.

a) In silico prediction of allele-specific splicing factor binding sites within OAS1 exon 3. Only the rs1131454-G allele creates a binding site for SFRS1 splicing factor; binding sites for SFRS2 are created by both alleles, with three or two sites in the presence of G or A allele, respectively. b) Experiment outline: description of allele-specific mini-genes with OAS1 exon 3 inserts, transfection in T24 and A549 cells, and splicing ratios of amplicons detected by RT-PCR with FP and RP primers. c) A representative agarose gel showing splicing events of mini-genes detected by RT-PCR in T24 and A549 cells; Vector corresponds to negative control; M corresponds to 100-bp size marker. Upper and lower bands correspond to Long and Short exon 3 splicing events with vector exon 1 (VE1). No alternative splicing event was identified between exon 3 insert and VE2 (data not shown). Each mini-gene was analyzed in three biological replicates and the results of one of two independent experiments are shown. d) The ratios of Long/Short OAS1 exon 3 expression quantified by densitometry of agarose gel bands. Splicing of Long exon 3 is significantly higher from the mini-gene with rs1131454-G allele compared to mini-gene with rs1131454-A allele. Fold changes (FC) were calculated from the splicing ratios. The dot plots are presented with means and SD; P-values are for unpaired, two-sided Student’s t-tests.