Figure 6: Strengthening weak splice sites relieves the LCR requirement in Dscam.

(a) Weak or suboptimal splice sites are built on sets of exon clusters in Dscam. The 5' and 3' ss motif scores were calculated using the splice site predictor²¹ (range is 0 to 1, with higher values predicting stronger splice sites). ‘*’ depicts the weak constitutive splice site downstream the docking site. ‘^*’ indicates splice sites that can be recognized as both 5′ and 3′ ss. (b) An overview of various mutant minigene constructs generated for the splicing assay for exon 6.47. A green arrow depicts activating the inclusion of the alternative exon; A red arrow depicts increasing the strength of the splice site. The wild-type and modified 5′ or 3′ splice site sequences are presented, with the scores on the right. Uppercase letters indicate exon sequences; lowercase letters indicate intron sequences. Mutated nucleotides are marked in red. (c) Effects of mutations on the inclusion of the alternative exon 6.47. Reverse transcription–PCR was performed to detect RNA splicing pattern. The band marked by ‘*’ is a nonspecific RT–PCR product. (d) Effects of mutations on the inclusion of alternative exon 6.47. (e) Overview of various mutant minigene constructs generated for splicing assay exon 6.48. Mutated nucleotides are marked in red. (f) Effects of mutations on the inclusion of the alternative exon 6.48. Reverse transcription–PCR was performed to detect RNA splicing pattern. (g) Effects of mutations on the inclusion of the alternative exon 6.48. Data are expressed as mean±s.d. from three independent experiments (d,g). WT, wild-type.