Figure 3.

SAP155 knockdown by siRNA reduces both FIR and P27Kip1 expression and increases their alternative splicing variants. A, SAP155 knockdown by siRNA (5 or 20 nmol/L for 48 or 72 hours) show reduced P27 expression. Some of the FIR protein is shifted to a lower molecular weight, indicating that SAP155 is required for FIR splicing. B, SAP155 knockdowns by siRNA for 48 hours inhibited P27 pre-mRNA splicing in a dose-dependent manner, as indicated by the P27*/P27 ratio. The P27*/P27 ratios were 0.21 (GL2 siRNA), 0.36 (2 nmol/L SAP155 siRNA), 0.48 (5 nmol/L), 0.58 (20 nmol/L), and 0.74 (50 nmol/L). When the P27*/P27 ratio for GL2 was set at 1, the P27*/P27 ratios were 1.7 (2 nmol/L SAP155 siRNA), 2.2 (5 nmol/L), 2.7 (20 nmol/L), and 3.5 (50 nmol/L). CyclinE expression also decreased with SAP155 siRNA silencing. C, FIR, cyclinE, p-cdk2, and cdk2 expression was suppressed, and RT-PCR shows that FIR splicing variants were increased by SAP155 knockdown by siRNA in HeLa cells. A novel FIR splicing variant, Δ5, was detected by RT-PCR. D, Cdk2 knockdown by siRNA decreased cyclinE expression but not FIR or SAP155 expression.