Salsa is required for splicing of the first intron of gurken mRNA. Germline-specific depletion of Salsa is associated with a significant increase in the retention of the first intron, but not of the second and third introns, of gurken mRNA. (A) RT-qPCR analysis of gurken transcript using intron–exon primers and an iScript cDNA library (random hexamer and oligo [dT] reaction mix). Germline-specific depletion of Salsa induced a significant retention of the first intron of gurken transcript, whereas the second and third introns were correctly spliced. Shown are the fold changes of intron retention. Relative normalized expression corresponds to values normalized with two distinct reference genes (β-actin and GAPDH) and relative to control conditions (mCherry RNAi). At least three biological replicates were used for each data set. Error bars indicate standard deviation. (B,C) Reverse transcription droplet digital PCR (RT-ddPCR) of gurken to accurately determine first intron unspliced/spliced ratio. Whereas in control ovaries (mCherry RNAi) the unspliced/spliced ratio of the first intron of gurken was only 0.06 ± 0.0072, after Salsa depletion (salsa RNAi-2) it raised to 0.33 ± 0.015. (B) Accurate quantification, by ddPCR, of spliced and unspliced isoforms of gurken first intron, relative to reference sequence: After Salsa depletion, there was an enrichment in unspliced intron 1 (unspliced t-test, P < 0.00001) and the corresponding decrease in intron 1 removal by splicing (spliced t-test, P = 0.011). (C) Graphic representation of the increase in gurken first intron ratio unspliced/spliced after Salsa depletion (salsa RNAi-2) (t-test, P < 0.00001). Two biological replicates were used for each RT-ddPCR data set. Error bars indicate standard deviation.
