Figure 7. VP1 inhibits Slicer activity of pre-assembled mature RISC.

(A) Schematic overview of the two experimental conditions of the Slicer assay designed to monitor the effect of recombinant (rec.) proteins on RISC assembly and Slicer activity (top) or on Slicer activity of pre-assembled RISC (bottom) (B) Slicer assays in Drosophila embryo lysates. RISC activity was analyzed in the presence of a non-targeting control siRNA (lane 1) or a specific Fluc siRNA (lane 2–10). Recombinant proteins were added before (lanes 3–6) or after (lanes 7–10) assembly of RISC as indicated. As a control for possible buffer effects, recombinant protein was substituted by protein storage buffer (lanes 1 and 2). (C) Western blot showing the endogenous AGO2 protein levels in embryo lysate after incubation for 2 hours with the indicated recombinant proteins. The blot was developed with AGO2 antibody 4D2. (D) Slicer assay using an siRNA with a 2′-O-methylated guide strand. A non-modified control siRNA (lane 1) or a Fluc siRNA duplex containing a 2′-O-methyl group at the 3′ terminal nucleotide of the guide strand (lanes 2–6) was added to embryo lysate 30 minutes prior to the addition of the indicated recombinant proteins. (E) Slicer assay using a 2′-O-methylated simplex guide RNA. A control siRNA duplex (lane 1) or a single-stranded Fluc specific guide strand with a 2′-O-methyl group at the 3′ terminal nucleotide (lane 2–6) was added prior to the addition of the indicated recombinant proteins. (F) Slicer assays in the presence or absence of ATP. Embryo lysate was incubated with a control siRNA (lanes 1 and 6) or a specific Fluc siRNA (lanes 2–5 and 7–10). ATP was then depleted (lanes 1–5) or depleted and subsequently regenerated (lanes 6–10) and Slicer activity was monitored. An asterisk (*) indicates a non-specific band appearing in RISC assays under ATP depleted conditions.