FIG. 3.

VA RNA-derived small RNAs are stably associated with RISC. A schematic diagram of RISC substrate RNAs is shown in panel A. The VA RNA genes were separated near the apical loop into two halves and cloned into a reporter mRNA, generating target regions complementary to the 5′ and 3′ halves of both VA RNAs (see reference 3). Immunopurified RISC from uninfected 293-Ago2 cells (Mock) or cells infected with wt900 was assayed for RISC activity against the synthetic reverse VA transcripts with target regions complementary to the 5′ or 3′ half of VA RNAI (B) or VA RNAII (C). Arrows indicate the span of the VA RNA target regions in respective transcript. The positions of 5′ and 3′ cleavage products generated by Dicer cleavage at the terminal stem in VA RNAI and VA RNAII are indicated by a dot. Bands labeled with * indicate the 3′ end cleavage product of the substrate RNA, which usually is degraded in S15 cytoplasmic extracts but is often seen in reactions using immunopurified RISC.