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. 2019 Jul 9;47(16):8746–8754. doi: 10.1093/nar/gkz572

Figure 1.

Figure 1.

Perturbing splicing of the recipient intron affects RPR maturation. (A) Split-RFP reporter. Act5C promoter (Pol II), grey; RFP exons, red; Drosophila virilis intron-encoding RPR, pink; splice sites (native), shown in lower case; Probe 1, RPR antisense probe. Intron, 701 nt; RPR, 353 nt. (See also Supplementary Table S1 for additional details on probes used). (B) Northern blot of RNA extracted from S2 cells treated with dsRNA to knockdown either lariat debranching enzyme (Ldbr KD) or GFP control (GFP KD) and transfected with the split-RFP reporter (Figure 1A). Asterisk, faster-migrating intermediate. Drosophila melanogaster (Dm) U6 snRNA, loading control for northern; RFP RT-PCR, RFP expression was used to assess transfection and splicing efficiency; GAPDH, loading control for RT-PCR. (C) Quantitation of northern blot in (B) shows that the level of Dv RPR is significantly decreased following Ldbr KD when compared to the control (GFP KD) (n = 3). (D) Schematic depicting the conserved location of RPR in the second intron of the ATPsynC gene in 12 sequenced Drosophila species. Box and whisker plot, showing donor (5′ splice-site, 5′ ss) and acceptor (3′ splice-site, 3′ ss) splice-site scores for the first and second intron in ATPsynC. Each dot represents the splice-site score for one of the 12 species (see also, Supplementary Figure S1D for scores in individual species). (E) Split-RFP reporter with ‘ideal splice-sites’. The split-RFP reporter was mutated to incorporate canonical (strong) D. melanogaster 5′ donor and 3′ acceptor splice sites. Mutations are depicted in red uppercase (see also Supplementary Figure S1D). (F) Northern blot of RNA extracted from S2 cells expressing D. virilis RPR from the reporter with native or ‘ideal splice-sites’ (A and E) (see also Supplementary Table S1). (G) Quantitation of the spliced-RFP mRNA in cells transfected with the ‘native’ (A) or ‘ideal splice-site’ (E) reporters. Gene expression was determined by RT-qPCR; RFP data normalized to GAPDH mRNA (n = 8). (H, I) Quantitation of Dv RPR expression in cells transfected with the ‘native’ (A) or ‘ideal’ (E) splice-site reporters; Dv RPR normalized to either Dm RPR (H) or RFP (I) expression (n = 8). Note: Significance values are P values, calculated by an unpaired t test. (G, H and I) or Wilcoxon's test (D): *, <0.05; **, <0.01; ***, <0.001. Error bars indicate standard deviation of the mean. (Supplementary Figure S1 and Supplementary Tables S1–S3 provide sequences of probes and primers.)