Figure 4.

Cleavage of the passenger strand of siRNA duplex by AGO2. (A) AGO2 immunopurified from siRNA-programmed S2 lysate under high-salt conditions was incubated with luc passenger siRNA radiolabeled at the 5′-end. The passenger strand is cleaved by AGO2 preloaded with luc guide siRNA. luc guide and passenger strand sequences are shown on the right. The cleavage site on the passenger is indicated with an arrow. (B) Detection of the cleaved passenger strand of siRNA duplex programmed in S2 lysates. Two sets of luc siRNA duplex (the nucleotide sequences are the same, but only passenger or guide strand of the duplex was radiolabeled at the 5′-end) were used. The passenger strand, but not guide siRNA being cleaved (9 nt) is detected. (C) In AGO2-lacking embryo lysate (AGO2⁴¹⁴ lysate) (Okamura et al. 2004), the cleaved passenger is not detected, as opposed to the wild-type embryo lysate, which indicates that cleavage of the passenger strand of the siRNA duplex occurs in an AGO2-dependent manner. The luc siRNA duplex (the passenger was radiolabeled at the 5′-end) was used.