The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells

Abstract

Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways.

FIGURE 1.

Armitage is localized in the Yb body in ovarian somatic cells. Shown is the immunofluorescence costaining of FLAG-Yb (red) and Armi (green) in a 5×FLAG-Yb transgene ovariole (A) and a stage 4 egg chamber (B–E). DNA was labeled with DAPI (blue). Armi was enriched in the nuage (Nu) in nurse cells (NC) and colocalized with Yb in somatic cells in the Yb body (YB). The inset in A is a magnified view of the boxed area in A. GSC, germ line stem cells; SCC, somatic cap cells; FC, follicle cell; Ge, germarium. S2, S3, and S5 designate stage 2, 3, and 5 egg chambers, respectively.

FIGURE 2.

Armi and Yb are in the same molecular complex. A, co-immunoprecipitation of Armi with FLAG-tagged Yb. The input was the ovarian lysates from FLAG-Yb transgenic and nontransgenic w¹¹¹⁸ flies, and the Yb complexes were co-immunoprecipitated with the anti-FLAG antibody. The upper panel was stained for Armi, which was present in both the transgenic and nontransgenic input as well as in the FLAG-immunoprecipitated complex of transgenic flies, but not in nontransgenic flies. The lower panel shows the same blot stained for FLAG-Yb. B, reciprocal co-immunoprecipitation of FLAG-Yb with Armi. The input was the ovarian lysate from FLAG-Yb transgenic females run on separate gels. The Armi-Yb complexes were co-immunoprecipitated with the anti-Armi antibody. The ovarian lysate of FLAG-Yb transgenic flies immunoprecipitated (IP) with beads but without antibody as a negative control. The left panels were stained for endogenous Armi, and the right panels were stained for FLAG-Yb.

FIGURE 3.

piRNA pathway components Armi and Piwi, but not Ago3 or Aub, are mislocalized in the Yb mutant. Shown is the immunofluorescence staining of Armi (A–C), Piwi (D–F), Ago3 (G–I), and Aub (J–L) in WT germaria and early-stage egg chambers (A, D, G, and J) and in Yb mutant germaria (B, E, H, and K) and early-stage egg chambers (C, F, I, and L). Armi and Piwi were mislocalized in somatic cells in the Yb mutant, whereas Ago3 and Aub were not affected. Ge, germarium; S2, stage 2 egg chambers.

FIGURE 4.

Yb is involved in the biogenesis of somatically expressed piRNAs. A, length distribution of small RNAs (excluding miRNAs) in WT and Yb mutant ovaries. The blue lines and red lines show the distribution of small RNAs in the WT and Yb mutant, respectively. Note that the level of 26–27-nt small RNAs that are enriched in Piwi-associated piRNAs was decreased in the Yb mutant. B, expression of piRNA cluster-derived small RNAs in the WT and Yb mutant. The relative expression levels of small RNAs derived from 17 previously reported piRNA clusters (clst.) are shown. Small RNAs uniquely mapped to these clusters were used for analysis. These clusters are grouped into three groups according to the expression preference of their piRNAs (19, 21, 22), with the germ line cell-enriched class colored in red, the intermediate class colored in orange, and the soma-enriched class colored in violet. The soma-enriched class clusters were significantly decreased in Yb mutant ovaries, the intermediate clusters were moderately decreased, and the germ line cell-enriched clusters were essentially unchanged. C, expression of transposon-derived small RNAs in the WT and Yb mutant. Shown are the relative expression levels of piRNAs generated by each transposon. Small RNAs were mapped to the consensus sequence of each transposon, allowing up to three mismatches. These transposons are grouped into three classes according to the expression preference of their piRNAs (21), with the germ line cell-enriched class colored in red, the intermediate class colored in orange, and the soma-enriched class colored in violet. The expression levels of piRNAs from the soma-enriched class were significantly decreased in Yb mutant ovaries, whereas those from the germ line cell-enriched class were essentially unchanged. D, piRNA densities distributed along the flamenco locus in Yb mutant and WT ovaries, with a map of the locus drawn to the scale corresponding precisely to the position of the x axis of the density charts. In addition, the map distinguishes plus-strand from minus-strand and marks the gypsy6 transposons in the flamenco locus in red and the non-gypsy6 transposons in black. Uniquely mapped small RNAs are plotted against the flamenco locus with a window size of 1 nt. The blue lines represent small RNAs produced from the plus-strand, whereas the red lines represent those produced from the minus-strand. The putative endo-siRNAs in the gypsy6 sublocus is enlarged in the upper inset, in which green bars and yellow bars represent 21-nt putative endo-siRNAs from the plus- and minus-strands, respectively. The lower inset shows the length profiles of flamenco-derived small RNAs in the WT (blue) and Yb mutant (red).

FIGURE 5.

Yb is involved in the biogenesis of flamenco-derived piRNAs and endo-siRNAs. A, nucleotide composition of flamenco-derived small RNAs from the WT and Yb mutant. Only small RNAs uniquely mapped to the flamenco locus were analyzed. B, size profiles of flamenco-derived small RNAs in the WT and Yb mutant. The blue curves and red curves represent small RNAs in WT and Yb mutant ovaries, respectively. C, relative expression levels of piRNAs and siRNAs from the flamenco locus in WT and Yb mutant flies. The ratio of the piRNA/siRNA expression level to miRNA expression level (with the WT level set as 1) is shown. Note the drastic decrease in the expression of both flamenco piRNAs and endo-siRNAs in the mutant. D, expression of putative piRNA precursors and small RNAs (including piRNAs and endo-siRNAs) in the WT and Yb mutant. The ratio of the expression level in the Yb mutant to that in the WT is shown. The expression level of putative piRNA precursors was normalized using the level of RP49. The positions and primer sequences analyzed here are found in Ref. . E, nucleotide composition of flamenco-derived 21 nt small RNAs from WT and Yb mutant ovaries. Only small RNAs uniquely mapped to the flamenco locus were analyzed.

FIGURE 6.

Somatically active transposons are desilenced in Yb mutants. Quantitative RT-PCR was performed to determine the expression of multiple transposons, relative to rp49, in WT (w¹¹¹⁸), Yb⁷²/FM6, and Yb mutant ovaries. Transcript levels from WT ovaries were set as 1, and -fold changes are indicated. The error bars indicate S.D.