SNP rs3736234 affects OLR1 AS pattern in both allelic series. (A) Schematic representation of the Low- and High-Risk minigenes used in this study, including the nucleotide identities and the relative position to the closest 3′ss (in base pairs) of the 6 SNPs in linkage disequilibrium in the two allelic series. pCMV indicates Cytomegalovirus promoter, SV40 refers to Simian Virus 40 polyadenylation signal, AMPr indicates ampicillin resistance cassette included in the vector backbone, coding exons are represented as rectangles, intronic sequences as thin lines, and 3′ UTR as thick line. Length in base pairs (bp) of exons, introns, and 3′ UTR is indicated by the numbers below. Arrows represent forward (splice junction) and reverse (vector-specific PT1 sequence) primers used for real-time PCR quantification of the exon 5 inclusion (INC) or skipping (SKIP) isoforms. (B) Exon 5 inclusion/skipping ratios of transcripts generated upon transfection of Low- and High-Risk minigenes in HeLa, HEK293-T, or SK-N-SK cells, and measured 24 h post-transfection. RT-qPCR values represent mean and standard deviations of 10 independent biological replicas. (C) Schematic representation of single point mutations introduced by site-directed mutagenesis in the OLR1 Low- and High-Risk minigenes to dissect the effect of each SNP in AS regulation. Nucleotide identities in the High-Risk series are indicated in bold, those in the Low-Risk series in normal lettering. (D,E) Quantification of inclusion/skipping ratios for the different mutants in C. Data represent mean and standard deviation of exon 5 inclusion/skipping ratios after normalization against Low-Risk (D) or High-Risk (E) minigenes for eight independent biological replicas. P-values obtained from Student's two-tailed heteroscedastic t-test comparing to Low- or High-Risk minigenes are indicated: (*) <0.05, (**) <0.01, (***) <0.001.
