Additional file 3: of Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5′ splice site strength

Altered splicing of transcripts encoding proteins required for mitotic progression after PRPF8 depletion. a RT-PCR analysis of RNA from PRPF8-depleted cells reveals a variety of splicing defects in transcripts that encode critical factors required for mitotic progression, including a retained intron (NUDC) and skipped exons (ASPM and SKA2). RTPCR analysis of ASPM, NUDC, and SKA2 using the primers indicated in the schematic are shown alongside the corresponding sashimi plots (control siRNA in black, PRPF8 siRNA in red). The splicing defects are indicated by arrowheads and counts spanning splice site junctions that change in the skipped exons are also indicated. b, c Splicing alterations in mitotic genes do not normally occur during transit through mitosis. Cal51 cells treated with nocodazole for 4 or 15 hours to induce mitotic arrest were monitored for mitotic index (b), and analyzed by qRT-PCR for splicing alterations previously observed following PRPF8 depletion (see Fig. 2) (c). Plots are relative to RNA levels in control siRNA-treated cells, assigned an arbitrary value of 1, and show the mean of triplicate readings from three independent experiments ± standard error of the mean (SEM). Mitotic index plots also show the mean from three independent experiments ± SEM. (PDF 264 kb