SRSF1 RRM1 and RRM2 are required for OLR1 AS regulation. (A) Schematic representation of the different SRSF1 protein expression mutants used in this experiment. OE indicates overexpression, WB stands for Western blot. (B–G) Real-time PCR quantification of the ratio of inclusion/skipping upon co-transfection of Low- or High-Risk minigenes with the indicated SRSF1 constructs in HeLa cells (+, empty vector; ++, 250 ng of protein expression vector; +++, 1000 ng of protein expression vector). In all cases, the total amount of transfected DNA was adjusted to 1000 ng with empty vector. Data represent mean and standard deviations of the ratio inclusion versus skipping after normalization by the ratio of the Low-Risk minigene for eight independent experiments. Protein overexpression was validated by Western blot using anti-T7 tag antibody (+, maximal amount of expression plasmid) and tubulin was used as loading control. Endogenous OLR1 isoforms were detected by semi-quantitative PCR. P-values obtained from Student's two-tailed heteroscedastic t-test are indicated: (**) <0.01, (***) <0.001.
