Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ("Slicer") activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as "Slicer" of the passenger strand of the siRNA duplex.[Keywords: RNAi; Argonaute; Slicer, RISC; Drosophila] Supplemental material is available at http://www.genesdev.org.

Argonaute proteins function in gene silencing induced by double-stranded RNA (dsRNA) in various organisms. In Drosophila, the Argonaute proteins AGO1 and AGO2 have been implicated in post-transcriptional gene-silencing (PTGS)/RNA interference (RNAi). In this study, we found that AGO1 and AGO2 depletion caused the accumulation of multicopied enhanced green fluorescence protein (EGFP) transgene transcripts in Drosophila S2 cells. Depletion of AGO1, the essential factor for miRNA biogenesis, led to an increased transcriptional rate of the transgenes. In contrast, depletion of AGO2, the essential factor for siRNA-directed RNAi, resulted in EGFP mRNA stabilization with concomitant shortening of the EGFP mRNA poly(A) tail. Our findings suggest that AGO1 and AGO2 mediate multicopied transgene silencing by different mechanisms. Intriguingly, Dicer2 depletion phenocopies AGO2 depletion, with an increase in EGFP protein levels and shortening of the EGFP mRNA poly(A) tail. The possibility that AGO2 and Dicer2 involve, at least in part, poly(A) length maintenance of transgene mRNA suggests a potentially important link between transgene silencing and poly(A) tails.