Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome

Abstract

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.

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

“…This endogenous small RNA defense mechanism shares many features with the RNAi mechanism first described in Caenorhabditis elegans (6). However, in some animals, a distinct small RNA defense mechanism has been described (7) using small RNAs that interact specifically with the PIWI clade of argonaute proteins (piRNA) (8)(9)(10)(11)(12). Production of piRNA is independent of Dicer enzymes (11) and, correspondingly, these small RNAs are slightly larger in size (24∼30 nt).…”

“…Despite recent progress, the mechanisms used by piRNA to promote silencing are not clear; evidence supporting both transcriptional and posttranscriptional silencing mechanisms has been reported (10,(18)(19)(20)(21). The fly genome codes for three PIWI family argonaute proteins used in the piRNA pathways are Piwi, Aub, and AGO3 (22).…”

“…The fly genome codes for three PIWI family argonaute proteins used in the piRNA pathways are Piwi, Aub, and AGO3 (22). Although Aub and AGO3 are restricted to the germ-line cytoplasm, Piwi localizes predominantly in the nucleus, while still present in the cytoplasm of both the germ line and the ovarian soma (8,10,23). Correspondingly, Piwi appears to be a key component of both germ line and somatic piRNA pathways (14,15).…”

“…Alternatively, Piwi could function directly in transposon silencing by using piRNAs. The majority of Drosophila piRNAs map to the pericentric or telomeric heterochromatin (8,10). In Schizosaccharomyces pombe, the RITS complex uses both an argonaute protein, Ago1, and an HP1 protein, Chp1, in targeting heterochromatin assembly (26).…”

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…As a slicer, Piwi can cleave targets [107,108]. Subsequently, the RNA products of this reaction may be loaded into the complementary Piwi-clade protein Ago3 for ping-pong amplification [107] (Box 1).…”