The docking site acts as an enhancer for long PGRP-LCa 3′ cassette selection. (A) Overview of the minigene constructs of D. melanogaster used to assess the effects of RNA pairing on splicing. Symbols used are the same as those in Figure 1. A series of constructs were generated by changing the docking site (CE1) context upstream of the 5′ intron-proximal splice site, albeit with similar RNA secondary structures to those of the WT. Predicted RNA pairing for the WT and a series of mutants (Ma1–Ma6, point mutations are shown in red) with the estimated equilibrium free energies (given in kcal/mol). (B) Effects of RNA pairing on LCx, LCy, and LCa inclusion according to mutation analysis. These mutations had little effect on the ratio of long to short cassette LCx and LCy, but significantly decreased and even switched the ratio of long to short cassette LCa. (C) Mutations within the docking site modulated the ratio of long to short cassette LCa. Quantitation of the data from B. Data are expressed as the percentage mean ± SD from three independent experiments. (D) Schematic diagrams of the constructs used to screen splicing proteins by RNA affinity purification and mass spectrometry. The sequences of the WT and mutated (Mu) single-stranded RNAs used for affinity chromatography, respectively, are shown. (E) Effects of protein knockdown on the ratio of long to short cassette LCa. PGRP-LC-M12, in which the selector sequences (CE1 and CE2) have been deleted, was used for cotransfection. These experiments revealed that the depletion of B52 resulted in a significant decrease in the ratio of long to short cassette LCa. (F) Quantitation of the data from E. Data are expressed as the percentage mean ± SD from three independent experiments. (*) P < 0.05.
