Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies

Abstract

Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. piRNAs are thought to derive from long transcripts spanning transposon-rich genomic loci and to direct an autoamplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce 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, Ago3-independent piRNA pathway centered on Piwi. 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 germline.

Figure 1

ago3 mutants. (A) The ago3 gene resides in pericentromeric heterochromatin on the left arm of chromosome 3. (B) ago3 mutant alleles were identified by TILLING. (C) Full-length Ago3 protein was not detected in trans-heterozygous ago3 ovaries, but was readily detected in heterozygotes and wild-type. (D) Protein levels in wild-type and ago3 ovaries, relative to ago3/TM6B (red line). The average ± standard deviation for at least three independent biological samples is shown. (E) Representative data for Aub, Piwi, Vasa, Argonaute1 (Ago1), Argonaute2 (Ago2), and Armitage (Armi) in ago3 ovaries. Tubulin served as a loading control.

Figure 2

Consequences of loss of Ago3 in the Drosophila germ line. (A) Mutually interdependent incorporation of Ago3 and Aub, into nuage in ovaries. Images correspond to single confocal sections (63× magnification); scale bar: 5 μm (Ago3, Aub, and Vasa panels) or 10 μm (Piwi). (B) and (C) Vasa localization within nuage is only partially congruent with that of Ago3 and Aub. Images correspond to single confocal sections of stage 4/5 egg chambers (63× lens, 4× zoom). Scale bars: 10 μm. (D) In 5−7 day old male adults, Vasa-expressing germ-line stem cells (GSC), which normally surround the somatic hub cells (hub), were not detected in ago3 testes. Since Vasa expression increases in ago3, the two images from ago3 mutants were acquired using reduced gain relative to wild-type and heterozygous testes. Scale bar: 10 μm (first three images) or 20 μm (fourth). (E) Stellate silencing in testes requires Ago3. Scale bars: 20 μm.

Figure 3

Aub- and Piwi-bound piRNAs disappear without Ago3. (A) Accumulation of Su(Ste) and AT-chX-1 piRNAs in testes required Ago3. (B) Accumulation of roo antisense piRNAs, as well as the AT-chX-1 piRNA, in ovaries required Ago3. (C) Accumulation of Piwi- and Aub-bound roo antisense piRNAs in the ovary was rescued in ago3 mutants by a single-copy transgene expressing Ago3 in the germ line (G) or in both the germ line and the soma (G+S). (D) Box plots illustrating the change in abundance of piRNAs, analyzed by transposon family, in ago3/TM6B versus Oregon R (left panel) and ago3 versus ago3/TM6B (right panel) ovaries. (E) Box plots illustrating the change in abundance for piRNAs for each of 95 transposon families, separated by group.

Figure 4

Three distinct groups of piRNAs. (A) The sense fraction of the piRNAs was compared for each transposon family between ago3 and ago3/TM6B ovaries. (B) An enlargement of the lower left corner of (A). Transposons labeled in red are present in the flamenco locus. Here and in (C) and (D), group III transposon families not present in flamenco are denoted with a red-filled black circle. (C) The sense fraction of piRNAs was compared between ago3 and Oregon R ovaries. (D) The sense fraction of the piRNAs was compared between aub and aub/CyO ovaries. (E) The normalized abundance of ping-pong pairs detected for all nine (ago3/TM6B) or four (ago3) possible PIWI protein pairings for the piRNA species uniquely associated with a single PIWI protein. Black bars, Bonferroni-corrected p-value < 0.005; gray bars, p-value > 0.005. p-values < 10^-30 are reported as p ∼ 0. Values are average ± two standard deviations.

Figure 5

Paradigmatic examples of each transposon group. (A) piRNA length distribution, abundance relative to consensus position, and ping-pong pair abundance. Blue, sense piRNAs; red, antisense. (B) Sequence-excess logos generated by subtracting the background from the relative frequency of each nucleotide at each position of the 23–29 nt RNAs in each sample. Only the nucleotide positions where foreground was significantly higher than background are shown (Bonferroni-corrected p-values < 0.001). Gray horizontal bars indicate the maximum possible value for each position. (C) Normalized and calibrated abundance of piRNAs uniquely associated with Ago3, Aub, or Piwi in ago3/TM6B or ago3 ovaries.

Figure 6

Loss of Ago3 increases expression of some group I and group III, but not group II, transposons. (A) Expression of group I, group II, and group III transposon families in ago3 ovaries, relative to wild-type (w¹¹¹⁸) ovaries, was assayed using whole-genome tiling microarrays. White bars: expression of the transposon family in both ago3/TM6B and ago3 ovaries was less than that of the 50^th percentile for expression of all mRNAs in wild-type, suggesting that expression change cannot be reliably quantified. Black bars: transposon families with expression greater than this threshold in one or both genotypes. Significant (false-discovery rate (FDR) < 0.02) and non-significant data (FDR > 0.02) are separated. (B) Quantitative RT-PCR was used to assess transposon expression, relative to Actin, for ago3 ovaries (black bars) and ovaries expressing one copy of a UAS-Ago3 transgene driven by nanos-Gal4 (gray bars) or actin5c-Gal4 (white bars), relative to ago3/TM6B ovaries.

Figure 7

A model for piRNA biogenesis. The Aub- and Ago3-dependent piRNA amplification cycle is envisioned to operate only in the germ line, whereas a Piwi-dependent, Aub- and Ago3-independent pathway is shown for somatic cells. In the germ line, Piwi can also partner with Ago3 to amplify piRNAs.