In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. We sequenced small RNAs associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide 10. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI.

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets.[Keywords: Piwi; retrotransposon; rasiRNA; Slicer; Drosophila] Supplemental material is available at http://www.genesdev.org.

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.