Figure 6: The retention of intron sequences immediately downstream of the last variable exon of each cassette.

(a) Schematic diagram of sDscamβ1 isoform expression. Symbols used are the same as in Figs 1 and 4. The expression of the specific combination of sDscam isoforms was achieved by alternative promoter activation, followed by alternative splicing. When sDscamβ1 was transcribed by a V5 promoter, both V5 and the downstream V6 cassette may have been spliced into the constant exon 5. The positions of the PCR primers are indicated. (b) Intron retention downstream of the 5′ splice site of the variable cassette (V5) in sDscamβ1 mRNA reads. Intron retention was much more abundant in the abdomen than in the cephalothorax. The 25-nt fragmented RNA-seq data sets were mapped to calculate the intron retention rate. Because of the low expression of the V5 and V6 isoform in the muscles, haemocytes and poison glands (Fig. 3c), the images of these RNA-seq reads are not shown. (c) RT–PCR analysis of V5 and V6 isoform expression. (d) Schematic diagrams of expression of sDscamβ2 isoforms. Different types of splice isoforms are indicated by the symbol "I, II, III, IV". (e) RT–PCR was used to detect isoform expression. These experiments revealed the splicing of multiple adjacent cassette variants. Due to the low expression of sDscam variable exons, nested PCR was necessary to amplify the products; only the primers used in the second PCR are depicted. The PCR products were confirmed by cloning and sequencing.