Competing RNA pairings were validated in Drosophila CG42235 pre-mRNA. (A) Overview of the 3′ variable region of Drosophila CG42235. Symbols used are the same as those in Figure 1. Introns are not drawn to scale. This gene contains five tandemly arrayed cassettes with five to eight exons at the 3′ variable region, which are alternatively spliced to the common exon 3. Based on the exon–exon junction reads from RNA-seq data, we calculated the splice isoforms. Value represents the number of exon–exon junctions/million reads. The 3′ splice site of exon 2 can be spliced only to the most proximal alternative 5′ splice site and the three most distal alternative 5′ splice sites (blue line), but not to two intermediate alternative 5′ splice sites (red line). In contrast, the 3′ splice site of exon 3 can be spliced only to the two most proximal alternative 5′ splice sites (blue line), but not to the three most distal alternative 5′ splice sites (red line). (B) Arrangement of cis intronic elements for RNA pairing interactions. For predicted intronic RNA pairing interactions, see Supplemental Figure S5. (C) Schematic diagrams of minigene constructs used to assess the effects of RNA secondary structure on 3′ alternative cassette selection. Mutations introduced into dsRNA are indicated on the left or right mutated sequences (M1, M2). Green arrow depicts activated inclusion of the proximal exon. (D) Effects of intronic RNA pairing on alternative cassette inclusion were validated by disruptive single mutations (M1, M2) and compensatory double mutations (M12). (E) Quantitation of the data in D. Data are expressed as the percentage mean ± SD. (*) P < 0.05, (**) P < 0.01. (F) Evolutionary shifts from the 3′ constitutive site to the alternative one are concomitant with the creation and expansion of RNA pairing. The origin and expansion of competing RNA pairings at the 3′ variable region during Drosophila evolution are shown. Cassette duplications are indicated by red squares and green circles, respectively.
