Figure 7.

DHX8 and Prp19 selectively regulate p21 mRNA splicing, and DHX8, like SKIP, is required for binding of U2AF65 to p21 unspliced mRNA. (A, left) qRT–PCR was used to monitor the ratio of unspliced to spliced p21, PUMA, or NOXA mRNAs. U2OS cells were transfected with control, SKIP, DHX8, or Prp19 siRNA for 48 h, and incubated in the presence or absence of etoposide (20 μM) for the indicated times. (Right panel, lanes 1–6) Protein lysates were subjected to immunoblot analysis. (B) RNA-IP analysis of binding of U2AF65 to p21 or PUMA unspliced or spliced mRNA. U2OS cells were transfected with control, SKIP, or DHX8 siRNA for 48 h. RNA samples were purified from nonprecipitated cellular lysates (input) or extracts precipitated with U2AF65 antibody. Immunoprecipitated p21 transcript was detected using qRT-PCR with the primers used in Figure 4E. Values are expressed as percentage of input RNA. Error bars represent the standard deviation obtained from three independent experiments. (C) Model for the role of SKIP in the regulation of p21 gene-specific splicing.