FIGURE 6.

NS3 inhibits siRNA-mediated target cleavage in the Drosophila embryo extract in vitro RNA silencing system. (A) In direct competition assays, RISC-mediated target RNA (0.5 nM) cleavage was induced by siRNAs (5 nM) and MBP–NS3 (0.4–755.0 nM) simultaneously added to Drosophila embryo extracts. (B) In indirect competition, RISC was preassembled by adding siRNAs (5 nM) to embryo extract for 30 min and target RNA (0.5 nM) and MBP–NS3 (0.4–755.0 nM) subsequently added. (C) For direct and indirect competition experiments the percentage of cleaved target is plotted as a function of the MBP–NS3 concentration relative to the percentage of cleaved target in the absence of MBP–NS3. (A,B) Lanes 1 include siRNAs and lack MBP–NS3; lanes 2 lack inducer siRNA and MBP–NS3. We note that Drosophila embryo extract was used at the same concentration as we used for RISC assembly experiments (1 μg/μL in the test tube).