Figure 3.

SKIP regulates p21 mRNA splicing in vivo. (A, top panel) Schematic diagram of the primer pairs used to detect p21 unspliced and spliced mRNAs. (Bottom panel) qRT–PCR analysis was used to determine the ratio of unspliced to spliced p21 mRNA. U2OS cells were transfected with control or SKIP siRNA, and incubated with etoposide as indicated. (B) The top panel shows a schematic representation of the primer pairs used to detect the PUMA, GADD45, or NOXA unspliced and spliced mRNAs, whereas the bottom panel shows the ratio of unspliced to spliced mRNA for each gene, as determined by qRT–PCR. U2OS cells were treated as in A. (C) U2OS cells were transfected with empty vector or pCMV-Flag-p21, and, 24 h later, were transfected with control or SKIP siRNA for another 48 h. Cells were left untreated or treated with etoposide (20 μM) for a further 18 h prior to Western blot analysis. (D) Rescue of SKIP knockdown with an siRNA-resistant vector. U2OS cells were transfected with control, wild-type, or mutant SKIP vectors, and, 12 h later, were transfected with control or SKIP siRNA prior to mRNA and protein analysis at 48 h.