Fig. 3.

StauC is required for processing of dsRNA to siRNA. (A) Newly emerged female L. decemlineata were injected with 1 µg GFP, StauC, Dicer-2a, R2D2, or Loqs dsRNA. Ten days after injection, the females were mated with male beetles. Freshly laid eggs were collected, embryonic extracts were prepared, and the protein concentration was determined. A mixture of lysate, ³²P-labeled dsRNA, and the reaction mixture containing ATP were incubated at 25 °C for 3 h. Then the reaction was deproteinized by adding proteinase K and the RNA was precipitated by adding 3 M sodium acetate and 3 volumes of absolute ethanol. The RNA was resolved on 16% acrylamide-urea gel. The gel was dried and analyzed using a phosphorImager. The first lane shows the labeled GFP as the marker for dsRNA. The arrows point to dsRNA complexes. (B) Embryonic lysate prepared as described in the legend of A, ³²P-labeled dsRNA and the reaction mixture containing ATP were incubated at 25 °C for 1 h. The RNA was resolved on nondenaturing 4% acrylamide gel. The gel was dried and analyzed using a phosphorImager. The first lane shows GFP dsRNA used as a marker, and the last lane shows dsRNA digested to siRNA with RNase III and used as a marker for siRNA. The arrows point to dsRNA and siRNA bands.