Figure 6.

PRMT5 depletion triggers Mdm4 alternative splicing and p53 activation in multiple organs. (A) Experimental strategy used to delete PRMT5 at mid-gestation (E10.5). Embryos were analyzed at E15.5 and E17.5. Upon TAM injection, no pups were born alive. (Bottom panel) Efficiency of CRE recombination taking place in different organs. (B) Weight of PRMT5 wild-type (EtOH) or PRMT5-deleted (TAM) whole embryos at E15.5 and E17.5. (Right panel) Representative example of E15.5 embryos with wild-type (left) or deleted PRMT5 (right). (C) Quantitative PCR (qPCR) quantification of p53 targets in the indicated organs upon PRMT5 deletion. (Bottom panel) PCR validation and relative quantification of the alternative splicing event taking place on the Mdm4 mRNA upon PRMT5 deletion in the same organs. (D) H&E staining of wild-type and knockout E15.5 lung and liver sections. In the liver, light-purple-stained hepatocytes and dark-purple-stained hematopoietic precursor cells are easily detectable. Note the dramatic loss of the latter and the corresponding loss of ki67 staining. Below each H&E staining are the IHC stainings of lung and liver sections from a representative embryo. CC3 was used to detect apoptotic cells, and Ki67 was used to detect proliferating cells. Mdm4 pre-mRNA senses defects in the spliceosomal machinery in cancer lines. (E) PCR quantification of the alternative splicing event taking place on the Mdm4 mRNA upon PRMT5 knockdown (KD). Scramble shRNA was used as a control (Scr). Treatment with the splicing inhibitor TG003 (or with DMSO vehicle control) was used as an alternative way of perturbing the splicing machinery. The experiments were performed in the indicated human cancer cells (shown at the top). GAPDH was used as a loading control. (F) Quantification of Mdm4fl/Mdm4s splicing levels 4 d after infection and 2-d selection in 1 mg/mL puromycin upon knockdown with three different shRNA lentiviral constructs (Sh1–Sh3) in U2OS cells. (Scr) Scrambled control shRNA.