A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors

Abstract

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.

Figure 1

Characterization of the Drosophila TUDOR proteins. (A) Cartoon showing all Drosophila melanogaster proteins containing TUDOR/SMN domains (blue boxes). All other significant protein domains identified via HHpred searches are indicated with coloured boxes and their identity is given to the right from N to C (ZnF: zinc finger; RRM: RNA recognition motif; BBC: B-Box C-terminal domain; DEAD: DEAD-Box RNA Helicase; Hel-C: Helicase C-terminal; HA2: Helicase associated domain; OB: oligo-nucleotide binding; CS: HSP20-like domain; DSRM: double-stranded RNA binding; TM: trans-membrane domain; KH: K homology; SNase: Staphylococcus nuclease; DUF: domain of unknown function; UBA: ubiquitin-associated domain). TUDOR proteins implicated in the piRNA pathway (including the ones from this study) marked with a black dot (left). The scale indicates amino-acid positions. The identified mouse orthologues (see Supplementary Figure S1), the number of identified TUDOR domains in fly (mouse) and the expression bias towards gonads in adult flies are shown to the right. Proteins with similar domain composition are grouped together. For CG14303, the ‘??’ indicate the non-annotated N-terminus. (B) The secondary structure cartoon (blue indicates β-strands, red α-helices) denotes the extended TUDOR domain and is based on Liu et al (2010a) (see also Supplementary Figure S1). The core TUDOR domain (SMART definition) is shown as an alignment for all identified TUDOR domains (‘e’ and ‘h’ above the alignment indicate β-strands and α-helices, respectively). The conserved Arginine and Aspartate residues present in all extended TUDOR domains are highlighted in green, aromatic cage residues in red, the Asparagine involved in sDMA binding in orange and a strongly conserved glycine in grey. To the left, the predicted likelihood of a domain to bind sDMA residues (based on the aromatic cage residues) is indicated with black (likely binder) and grey (potential binder) circles.

Figure 2

The set of TUDOR proteins involved in the Drosophila piRNA pathway. (A) Cartoon of a Drosophila ovariole (somatic cells are in green, germline cells are in beige). The RNAi systems used for the two cell types are listed. (B) Immunostaining of Armitage (green) and DNA (blue) in egg chambers expressing RNAi constructs in a tissue-specific manner (left: wild type; middle: soma knockdown via tj-GAL4>hpRNA; right: germline knockdown via MTD-GAL4>shRNA or NGT-GAL4>Dcr-2+hpRNA). Monochrome panels show only the anti-Armitage channel. (C) Bright field images of ovarioles stained for β-GAL activity. The individual genotypes represent soma-specific knockdowns of the indicated genes in the background of the gypsy-lacZ sensor described in Sarot et al (2004). zucchini knockdown serves as a positive control and spindle-E as negative control. Of all TUDOR knockdowns, only those against CG4771 or Yb resulted in sensor de-repression. (D) Changes in steady-state levels of HeT-A and blood transposon transcripts upon knockdown of individual TUDOR proteins in the germline with the shRNA (black/gray) or the hpRNA (red/rose) knockdown systems (normalized to no-hairpin controls via rp49; log scale; n=3; error bars indicate s.d.). Identity of knocked down genes identical to the legend in (E). (E) Fertility rates of females with germline-specific knockdown of indicated TUDOR proteins using the shRNA (black) and the hpRNA (red) systems (∼200 eggs per experiment; n=3; error bars indicate s.d.).

Figure 3

Vreteno is a novel piRNA pathway member. (A) Overview of the CG4771 (vreteno) genomic locus indicating flanking genes (blue), the HP36220-insertion site (pink triangle) and the extent of the genomic rescue construct. (B) Cartoon of the CG4771 protein domain structure and sequence alignment of the C-terminal TUDOR domain in distantly related Drosophilids (virilis, mojavensis, grimshawi, willistoni, melanogaster, pseudoobscura). Aromatic cage residues and the conserved Arg/Asp residues colour coded as in Figure 1B. (C) Changes in steady-state transposon levels (n=3; s.d.) upon CG4771 knockdown (normalized to no-hairpin controls) in soma (green) or germline (beige) in comparison to those in CG4771[HP36220] mutants (black; normalized to heterozygotes). (D) Immunostaining of Piwi in wild-type and CG4771[HP36220] mutant egg chambers. (E) The occasionally observed egg chamber morphology of CG4771[Δ1] (vreteno) mutants, which originally led us to name the gene ‘avocado’ (DNA stained with DAPI). (F) RNA levels of CG4771, of the flanking genes HP1c and CG6985 and of actin-5C in vreteno[Δ1] mutant ovaries compared with vreteno[Δ1]; GFP–vreteno rescued ovaries (values normalized to w[1118] controls). (G) Immunostaining of Piwi in vreteno[Δ1] mutant egg chambers and in vreteno[Δ1] mutant egg chambers expressing a GFP–vreteno rescue construct. (H) Steady-state RNA levels of the HeT-A, blood and ZAM transposons in vreteno[Δ1] mutant ovaries compared with vreteno[Δ1]; EGFP–vreteno rescued ovaries (values normalized to heterozygous siblings; n=3; error bars indicate s.d.). (I) Immunostaining of Vreteno in wild-type and vreteno[Δ1] mutant egg chambers at identical microscope settings.

Figure 4

Vreteno is essential for primary piRNA biogenesis in the soma. (A) Immunostaining of Piwi (lower panels) in wild-type egg chambers (left) in comparison to egg chambers expressing hpRNAs against armitage (centre) or vreteno (right) specifically in somatic cells. Armitage and Vreteno stainings indicate the knockdown efficiency. (B) Normalized piRNA profiles (23–30 nt small RNAs) obtained from control ovaries (vret heterozygote; black) in comparison to profiles obtained from indicated mutant ovaries (red) mapping uniquely to the soma-specific piRNA cluster flamenco. The y axis for the heterozygote plot is representative for all plots.

Figure 5

Vreteno is essential for piRNA biogenesis in the germline. (A) Immunostaining of Piwi, Aubergine and Ago3 in wild-type egg chambers (top row) in comparison to egg chambers expressing shRNAs against armitage (centre row) or vreteno (lower row) specifically in germline cells. Armitage and Vreteno stainings indicate the knockdown efficiency. (B) Normalized piRNA profiles (23–30 nt small RNAs) obtained from control ovaries (vret heterozygote; black) in comparison to profiles obtained from indicated mutant ovaries (red) mapping uniquely to the germline-specific piRNA cluster 42AB (sense and antisense piRNAs are indicated with peaks pointing up- and downwards). (C) Normalized piRNA profiles obtained from control ovaries (black) in comparison to profiles obtained from indicated mutant ovaries (red) mapping to the germline-dominant Rt1b transposon.

Figure 6

Vreteno, Zucchini and Armitage are essential primary piRNA biogenesis factors but are dispensable for the ping-pong cycle. (A) Length profiles of all repeat-derived (transposon and satellite repeats) small RNAs (18–30 nt) isolated from ovaries of the indicated mutants and their respective heterozygous controls (all heterozygote libraries normalized to 1 million repeat-derived 23–30 nt RNAs). Sense populations are in blue and antisense populations are in red. The fold decrease in the respective populations (23–30 nt only) is indicated. (B) Bar diagram indicating the changes of normalized piRNAs mapping antisense to the indicated transposons (left) in the indicated mutant ovaries compared with the respective heterozygous control ovaries (het/mut ratios are given as log2 values). Grey bars indicate values below 1 (less than two-fold changes). Identity of the analysed mutants is given at the bottom. Transposons are grouped into germline-dominant (red), intermediate (yellow) and soma-dominant (green) based on Malone et al (2009). The heatmaps indicate degree of maternal piRNA inheritance (yellow: strong; red: weak) and ping-pong signature (blue: strong; white: absent) of each individual element. (C) Scatter plots of the log2 values plotted in (B), where individual transposons are colour coded as in (B). (D) Ping-pong signatures of the individual transposons (classification and order as in (B) in the average heterozygote (het.) and the indicated mutants as a heatmap ranging from strong signals (dark blue) to no signal (white). The F-element is indicated with an arrow. (E) Normalized piRNA profiles (sense and antisense) mapping to the F-element. Compared are populations from heterozygotes (black) to the indicated mutants (red). Ping-pong signatures (basis for the heatmap in (D) are shown to the right of each plot.

Figure 7

Vreteno is a novel Yb-body component. (A) Subcellular localization of GFP-tagged Vreteno or Armitage (optical section of egg chambers) expressed under the respective endogenous regulatory regions. (B) Confocal section through the follicular epithelium of a GFP–Vreteno (green) expressing egg chamber stained for Armitage (red) and DNA (blue). (Right panel) Merge of all three channels (co-localization of Vreteno and Armitage results in yellow). (C) Immunostaining of Vreteno (green), Piwi (red) and DNA (blue) in egg chambers, where clones of cells mutant for the indicated genes (left) have been induced in the follicular epithelium (clone borders are indicated with a yellow line). (D) Co-immunostaining of Vreteno (left) and Armitage (right) in OSCs transfected with siRNAs against EGFP (top), Yb (middle) or zucchini (bottom). (E) Co-IPs of Armitage and Vreteno from OSC lysate. Western blots against Armitage, Vreteno, Piwi and Yb. For the Armitage western blot (Armi-IP), only 1/15th compared with all other blots was loaded in the IP-lanes. Beads lacking antibody (IP-beads) served as control.

Figure 8

The Tdrd12 orthologues CG11133 and CG31755 are essential primary piRNA pathway factors in the germline. (A) Cartoon overview of the Tdrd12 proteins in fly and mouse (Tudor domains: blue; other domains as indicated). (B) Immunofluorescence staining for Armitage and GFP-tagged Yb, CG31755 or CG11133. (Lower panels) Merge of the three channels (DNA, GFP, Armitage). (C) The left chart shows changes in steady-state levels of the mdg1 transposon upon knockdown of CG11133, CG31755 or both together in OSCs. Values were normalized to GFP control knockdowns via rp49. The right chart indicates the efficiencies of the respective knockdowns (n=3; error bars indicate s.d.). (D) The left chart shows changes in steady-state levels of the HeT-A and blood transposons upon knockdown of CG11133, CG31755 or both together in germline cells (MTD-GAL4>shRNA). Values were normalized to ‘no-hairpin’ control flies via rp49. The right chart indicates the efficiencies of the respective knockdowns (n=3; error bars indicate s.d.). (E) Immunofluorescence staining for Piwi in wild-type egg chambers (left) compared with egg chambers where the indicated genes have been knocked down via shRNAs in the germline only. For the CG31755 and CG11133 single knockdowns, most egg chambers displayed a wild-type Piwi pattern (top), while 3–5% displayed the phenotypes of the lower panels. The phenotype for the double knockdown was fully penetrant.