Figure 4.
GW182 silences the expression of bound transcripts. (A) Schematic representation of the F-Luc-5BoxB tethering reporter and of the F-Luc reporter control. (B,C) S2 cells were transfected with the F-Luc-5BoxB reporter or the F-Luc control, a plasmid expressing Renilla luciferase, and vectors expressing the λN-peptide or λN-GW182. Firefly luciferase activity was normalized to that of Renilla and set to 100 in cells expressing the λN-peptide alone. Mean values ± standard deviations from four independent experiments (n = 4) are shown. In C, the corresponding RNA samples were analyzed by Northern blot. (D) F-Luc-5BoxB or F-Luc mRNA levels were quantitated and normalized to the R-Luc transfection control in four independent experiments, including that shown in C. Normalized F-Luc mRNA levels in cells expressing the λN peptide alone were set to 100%. Mean values are shown. Error bars represent standard deviations. (E) The normalized values of firefly luciferase activity shown in B were divided by the normalized mRNA levels shown in D to estimate the net effect of tethering GW182 on protein synthesis. (F,G) S2 cells were transfected with plasmids for the λN-tethering assay. Cells were harvested at the indicated time points after addition of actinomycin D. The decay of the F-Luc-5BoxB mRNA was monitored in cells expressing the λN-peptide (F) or λN-GW182 (G). (H) The levels of the F-Luc-5BoxB mRNA normalized to rp49 mRNA in three independent experiments are plotted against time. mRNA half-lives (t1/2) calculated from the decay curves are indicated.