Fig. 4

Loss of prp19 or plrg1 phenocopies, loss of ccdc94.

(A) Wild-type embryos were injected with the indicated morpholinos. RNA was harvested at 30 hpf, reverse transcribed using oligo-dT primers and analyzed for the expression of p53 mRNA by qPCR. Expression of the gapdh gene was also analyzed to normalize p53 mRNA levels. All values were then compared to the average value from uninjected embryos, which was adjusted to one. (B) RNA from (A) was reverse transcribed with random hexamers, and intron 9 of the p53 gene was analyzed by qPCR to determine levels of p53 pre-mRNA. Expression of 28S RNA was also analyzed to normalize p53 pre-mRNA levels. All values were then compared to the average value from uninjected embryos, which was adjusted to one. (C, D) Siblings and mutants from the plrg1(hi3174aTg) line were distinguished by morphology at 30 hpf and collected for analysis. RNA was harvested and analyzed by qPCR as in (A, B). For panels (A–D), error bars represent the standard error of the mean from at least three independent experiments. (E) Wild-type embryos were injected with the indicated morpholinos and analyzed at 30 hpf for p53 protein similar to Figure 3E. (F) Wild-type embryos were injected with the indicated morpholinos, irradiated (or not) at 24 hpf with 8 Gy and analyzed three hours later by whole-mount immunofluorescence to detect activated Caspase-3. Three independent experiments showed that knockdown of prp19 or plrg1 radiosensitized the embryos. (G) Activated-Caspase-3-specific immunofluorescence from (F) was quantified as in Figure 1D. (H) Genetic diagram showing that the Ccdc94 and core members of the Prp19 complex inhibit the transcription of p53, and therefore p53-mediated induction of puma expression, and normally restrict IR-induced mitochondrial apoptosis, upstream of Bcl-2. For all relevant panels in this figure, morpholinos were injected at the same concentrations as described in Figure 4B-4D. RT; reverse transcriptase, mm; mismatch, ccdc; ccdc94, prp; prp19. See also Figure S8.