Antagonistic effects of SRSF2 and SRSF1 in the regulation of OLR1 exon 5 AS. (A) Schematic representation of the deletion mutants of predicted binding sites for SRSF2 and SRSF1. Predicted binding sites for SRSF2 and SRSF1 in the region of rs3736234 are indicated as gray and black boxes, respectively. The different nucleotide variants in rs3736234, as well as the sequence deletions introduced in the constructs, are also indicated. (B,C) Effects of deletion of putative SRSF2/SRSF1-binding sites on OLR1 exon 5 AS. Ratio of exon 5 inclusion/skipping was determined for the indicated constructs as in previous figures. Data represent mean and standard deviations after normalization by the results of the Low-Risk minigene for four independent experiments. (D,E) Quantification of splicing changes for the Low- and High-Risk minigenes upon SRSF2 (D) or SRSF1 (E) overexpression. Cells were co-transfected for 48 h with the indicated minigenes and the corresponding protein expression vector (numbers indicate ng of expression vector utilized). Total amounts of transfected plasmids were raised to 1000 ng by using pCMV vector. Data represent mean and standard deviation for eight independent biological experiments. Insets show protein levels for the control and the maximum overexpression condition detected with anti-T7 tag antibodies. (F,G) Quantification of splicing changes for the Low- and High-Risk minigenes upon SRSF2 (F) or SRSF1 (G) knock down by RNAi. Numbers below indicate the final concentration of siRNAs used for each of the conditions. Insets show the RNA (RT-PCR, SRSF2) or protein (western blot, SRSF1) levels in scramble siRNA and SRSF2/SRSF1 siRNA knockdown conditions. (+) 100 µM. Values represent mean and standard deviations for six independent biological replicas. P-values obtained from Student's two-tailed heteroscedastic t-test used in this figure are indicated: (*) <0.05, (**) <0.01, (***) <0.001.
