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. 2006 Jul 15;20(14):1885–1898. doi: 10.1101/gad.1424106

Figure 7.

Figure 7.

Endogenous miRNA targets are degraded by deadenylation and decapping. (A) S2 cells depleted of GFP, AGO1, GW182, CAF1, NOT1, or the DCP1:DCP2 decapping complex were transfected with the F-Luc-Vha68-1 reporter. Firefly luciferase activity and the corresponding mRNA levels were measured and analyzed as described in Figure 6, A and B. (B) Expression levels of endogenous Vha68-1 mRNA in cells depleted of AGO1, GW182, CAF1, or DCP1:DCP2. The signals from the Northern blot were normalized to rp49 mRNA (not shown). These values are compared with the values measured by microarray. Values are given as fold changes relative to the values obtained cells treated with GFP dsRNA. (n.d.) Not determined. (C) RNA samples shown in B, lanes corresponding to the control and the DCP1 + 2 knockdown, were treated with RNase H in the absence or presence of oligo(dT) and analyzed by Northern blot. (D,E) The decay of Vha68-1 and Axs mRNAs was monitored in depleted cells following inhibition of transcription by actinomycin D. The levels of the Vha68-1 and Axs mRNAs were normalized to rp49 mRNA and plotted against time (not shown for Vha68-1 mRNA). mRNA half-lives (t1/2) calculated from the decay curves are indicated.