Similar bidirectional pairings were experimentally confirmed in exon cluster 4 of hymenopteran Dscam. (A) Overview of the deleted minigene constructs of Apis mellifera. Symbols used are the same as those in Figure 1. The nucleotides with greatest identity to the dual docking sites and selector sequences are shown in different colors. (B) The predicted RNA pairings of Dscam pre-mRNA. Mutations introduced into dsRNA are indicated on the left or right mutated sequences (M1–M8). The green arrow depicts activation of the inclusion of the proximal exon, while the dashed green arrow depicts the potential activation. (C) Effects of bidirectional RNA pairings on exon 4 inclusion are indicated for disruptive single mutations (M1–M8) and compensatory double mutations (M12; M34; M56; M78). The band marked by “*” is a nonspecific RT-PCR product. (D) Effects of RNA pairings on exon 4.1 inclusion. The PCR lanes represent the single exon 4-containing band cut from C. Exon 4.1-specific restriction digestion was performed to analyze the frequency of exon 4.1 utilization. (E) Quantization of the data in left panel of D. Data are expressed as percentages of the mean ± SD from three independent experiments. (***) P < 0.001 (Student's t-test, two-tailed). (F) Exon 4.1 inclusion negatively correlated with the strength of upstream RNA pairing. (G) Quantization of the data in the right panel of D. (H) Exon 4.1 inclusion positively correlated with the strength of downstream RNA pairing.
