FIGURE 4.
Dicer-2 interacts with Wispy. (A) Interaction of endogenous Wispy and Dicer-2 was assessed by coimmunoprecipitation from 90 min embryo extracts (wt). dcr-2L811fsx null embryo extracts (dcr2−) or immunoprecipitation with nonspecific IgGs were used as negative controls. The pellets were treated (+) or not (−) with 10 µg of RNase A. Inputs are shown on the left. UNR was included as a specificity control. The asterisk denotes a background band. (B) Dicer-2 interacts with the carboxy-terminal domain of Wispy. (Left) Schematic representation of Wispy and the fragments used in the experiment. (NT2) Nucleotidyl-transferase 2 domain; (PAD) poly(A) polymerase-associated domain. Numbers above Wispy indicate amino acid positions. (Right) Recombinant Dicer-2 was mixed with 35S-labeled Wispy fragments 1–5, and immunoprecipitated using affinity-purified antibodies. Firefly luciferase (Luc) was used as negative control. Wispy inputs (10%) and pellets (30%) are shown in the upper and middle panels, respectively. The lower panel shows the immunoprecipitated Dcr-2. (C) Ago-2, but not R2D2, interacts with Wispy. Immunoprecipitation of Wispy was performed as in A, and the presence of Ago-2, R2D2, and Dicer-2 in the pellet assessed by western blot. Tubulin was used as background control. i, 10% input.