FIG. 8.

Early poly(A) signals are more sensitive to Pcf11 depletion. (A) Northern blot analysis of total RNA extracted from Pcf11-depleted S2 cells transfected with Adh-UTR-9-P1, Adh-UTR-9-P2, or Adh-UTR-9-P3 as described for Fig. 3. Mock experiments involved cells not treated with Pcf11 dsRNA (lanes 1 to 3); the Adh probe was that shown in Fig. 2, and the 18S rRNA was that described for Fig. 7. (B) Band intensity quantification of the P2/readthrough and P3/readthrough transcript ratios detected by Northern blotting, with error bars based on two independent experiments. (C) Real-time RT-PCR quantification of Pcf11 mRNA in cells treated with dsRNA relative to control; Pcf11 mRNA levels were normalized to that of Rpl32. (D) Northern blot analysis of total RNA from S2 cells depleted of Cdk9, CycT, or Cdk9 and CycT and transfected with the same constructs as in panel A. (E) Real-time RT-PCR quantification of Cdk9 and CycT mRNA depletion, as described for panel C. (F) Northern blot analysis of total RNA from Fcp1-depleted S2 cells transfected with the same constructs as shown above. (G) P2/readthrough and P3/readthrough band intensity ratios detected by Northern blotting; error bars are based on two experiments. (H) Real-time RT-PCR quantification of the levels of Fcp1 mRNA in cells depleted via RNAi.