Fig. 1.

IRAS strategy illustrated by dual-fluorescence reporters. (A) Two complementary reporters differ in several nucleotides. Reporter #1 translates GFP when the alternative exon is included and RFP when the alternative exon is skipped. Reporter #2 translates RFP from the included isoform and GFP from the skipped isoform. Therefore, the ratios of GFP/RFP or RFP/GFP represent the inclusion ratio of the alternative exon in reporters #1 and #2, respectively. Factors that alter these ratios without affecting the inclusion ratio are false positives in screens. Possible actions of false positives that increase GFP/RFP ratio are labeled in purple (dark gray in the print version). False positives that increase RFP/GFP ratio are not drawn. (B and C) Expected phenotypes induced by true splicing activators and repressors and false positives in screens using cells stably express either reporters #1 or #2. A true splicing regulator changes GFP/RFP ratios in the two reporter cells in the opposite direction, whereas a false positive more likely affects the GFP/RFP ratios in the same direction. False positives that increase RFP/GFP ratio are not drawn.