Collapse of Germline piRNAs in the Absence of Argonaute3 Reveals Somatic piRNAs in Flies

Abstract

Summary Piwi-interacting RNAs (piRNAs) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, “piRNA clusters.” piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second piRNA pathway centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.

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

“…S2), we next looked at the impact of germ-line Piwi depletion on the localization pattern of Aub. Mutations disrupting the ping-pong amplification process can lead to mislocalization of Aub from the peri-nuclear structure nuage (13,34), the proposed site of secondary piRNA production (34). Similar to earlier observations using mutants that disrupt Piwi in both germ line and soma (14), we found no impact on Aub localization to the nuage (Fig.…”

“…Telomeric retrotransposon HeT-A and LTR retrotransposon Burdock show the most dramatic effects. In general, transposons that show increased expression in germ-line Piwi knockdown lines were the same as those that showed increased expression in an ago3 mutant line as reported in Li et al (13) (Table 1). This correlation argues that germ-line Piwi functions in the same pathway as Aub and AGO3 (8,9).…”

“…3A). However, mutations in piRNA pathway components can lead to mislocalization of Piwi protein (14,36); aub QC42 /aub HN2 results in a strong decrease in the level of Piwi in the nucleus (13). Here, immunofluorescent staining experiments show a notable depletion of Piwi signal in the germline nuclei of Aub knockdown ovaries (Fig.…”

“…This correlation argues that germ-line Piwi functions in the same pathway as Aub and AGO3 (8,9). However, three cases clearly do not follow this pattern: Transposons Max, Idefix, and Invader1 are significantly up-regulated in Piwi germ-line-knockdown ovaries (Table 1), but are reported to show little to no response to an ago3 mutation (13). This discrepancy suggests an additional role for Piwi.…”

“…In the Drosophila melanogaster female gonad, two distinct piRNA pathways have been identified that drive transposon silencing in the germ line and the soma, respectively (13)(14)(15). In the germ line, piRNA biogenesis involves both primary processing and a secondary amplification pathway (ping-pong amplification) (8,9,16), whereas piRNAs in the soma are generated solely from primary transcripts (primary pathway) (14,15,17).…”