FIGURE 6.

Dicer-2 promotes polyadenylation of r2d2 mRNA. (A) R2D2 protein levels decrease in dcr-2 null flies. Protein levels were assessed by western blot. Hrp48 was used as loading control. The asterisk denotes a nonspecific band. (B) Dicer-2 binds to endogenous r2d2 mRNA. Dicer-2 was immunoprecipitated from 90 min embryo extracts and the presence of r2d2 mRNA in the pellet assessed by RT-PCR. M, marker; i, input. (C) r2d2 3′ UTR promotes translation in a polyadenylation-dependent manner. The translation efficiency of a luciferase reporter containing the 3′ UTR of r2d2 (r2d2-A₀) was compared with reporters lacking r2d2 and either containing (A+) or not (A₀) a poly(A) tail. An r2d2 construct with cordycepin at its 3′ end was included (r2d2-A₀-co). Translation was assessed as described in the legend of Figure 5B. The plot represents the average ± STDEV of three experiments. Statistical significance was analyzed by unpaired Student's t-test. Cordycepin prevents translation of r2d2, suggesting that the reporter acquires a poly(A) tail during the reaction. (D) r2d2 3′ UTR is polyadenylated in embryo extracts, and Dicer-2 depletion reduces the polyadenylation efficiency. Assays were performed as described in the legend of Figure 2B. (E) Polyadenylation of r2d2 mRNA is reduced in dcr-2 and wispy, but not in ago2 null embryos. PAT assays were performed in wt (w¹¹¹⁸), dcr-2^L811fsX, wisp^KG5287/Df, and ago2⁴¹⁴ embryos as indicated in the legend of Figure 2A. To increase the sensitivity of the assay in dcr-2 extracts, amplified products were visualized by Southern blot using an r2d2-specific probe (upper left panel). To ensure that r2d2 mRNA was present in null embryos, semi-quantitative PCR with oligonucleotides detecting r2d2 was performed from the same RNA samples used for the PAT assay (lower panels).