A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors
2011; Springer Nature; Volume: 30; Issue: 19 Linguagem: Inglês
10.1038/emboj.2011.308
ISSN1460-2075
AutoresDominik Handler, Daniel Olivieri, Maria Novatchkova, Franz Gruber, Katharina Meixner, Karl Mechtler, Alexander Stark, Ravi Sachidanandam, Julius Brennecke,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle23 August 2011free access A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors Dominik Handler Dominik Handler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Daniel Olivieri Daniel Olivieri Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Maria Novatchkova Maria Novatchkova Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Franz Sebastian Gruber Franz Sebastian Gruber Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Katharina Meixner Katharina Meixner Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Karl Mechtler Karl Mechtler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Alexander Stark Alexander Stark Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Ravi Sachidanandam Ravi Sachidanandam Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Julius Brennecke Corresponding Author Julius Brennecke Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Dominik Handler Dominik Handler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Daniel Olivieri Daniel Olivieri Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Maria Novatchkova Maria Novatchkova Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Franz Sebastian Gruber Franz Sebastian Gruber Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Katharina Meixner Katharina Meixner Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Karl Mechtler Karl Mechtler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Alexander Stark Alexander Stark Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Ravi Sachidanandam Ravi Sachidanandam Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Julius Brennecke Corresponding Author Julius Brennecke Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Author Information Dominik Handler1, Daniel Olivieri1, Maria Novatchkova1,2, Franz Sebastian Gruber1, Katharina Meixner1, Karl Mechtler1,2, Alexander Stark2, Ravi Sachidanandam3 and Julius Brennecke 1 1Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria 2Institute of Molecular Pathology (IMP), Vienna, Austria 3Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA *Corresponding author. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr Bohrgasse 3, Vienna 1030, Austria. Tel.: +43 179 044 4508; Fax: +43 179 044 110; E-mail: [email protected] The EMBO Journal (2011)30:3977-3993https://doi.org/10.1038/emboj.2011.308 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The PIWI-interacting RNA (piRNA) pathway is an animal-specific small RNA pathway that silences selfish genetic elements such as transposons in gonads (Malone and Hannon, 2009; Khurana and Theurkauf, 2010; Senti and Brennecke, 2010). At the core of this pathway act Argonaute proteins from the PIWI clade and their bound small RNAs, generally referred to as piRNAs. Mutations in PIWI proteins or in factors involved in piRNA biogenesis or piRNA-mediated silencing lead to de-silencing of transposons, to widespread DNA damage and ultimately result in sterility. The analyses of piRNA populations from vertebrates and invertebrates have provided genuine insight into the genomic origin of piRNAs (Aravin et al, 2006, 2007, 2008; Girard et al, 2006; Lau et al, 2006; Vagin et al, 2006; Brennecke et al, 2007; Li et al, 2009; Malone et al, 2009; Robine et al, 2009; Saito et al, 2009). The three major piRNA sources are long RNAs originating from discrete genomic loci typically enriched in transposon sequences (piRNA clusters), transcripts from active transposons and finally mRNAs from numerous endogenous genes. The genetic and mechanistic principles of piRNA biogenesis are only poorly understood but sequence analyses of piRNA populations indicated that two modes of piRNA biogenesis exist (reviewed in Senti and Brennecke, 2010). On the one hand, during primary piRNA biogenesis presumably single-stranded precursor transcripts are processed in a seemingly random manner into 23–30 nt primary piRNAs (Lau et al, 2009; Li et al, 2009; Malone et al, 2009; Saito et al, 2009). On the other hand, transposon sense transcripts (typically from active elements) and antisense transcripts (typically from piRNA clusters) participate in the process of secondary piRNA biogenesis: Here, piRNA-mediated cleavage of the target transcript triggers the production of a novel piRNA with the reciprocal polarity (Brennecke et al, 2007; Gunawardane et al, 2007). Hallmarks of this so-called ping-pong amplification of piRNAs are conserved from sponges to mammals (Aravin et al, 2007; Grimson et al, 2008). The existence of two distinct piRNA biogenesis branches is particularly evident in the Drosophila ovary. Within ovarian germ cells, the three PIWI proteins Piwi, Aubergine and Argonaute 3 (Ago3) are co-expressed and piRNAs are generated via the primary and secondary pathways. The two major players of the secondary ping-pong pathway are Aubergine and Ago3 with Aubergine binding primarily cluster derived antisense piRNAs, while Ago3 is primarily complexed with transposon mRNA-derived sense piRNAs (Brennecke et al, 2007; Gunawardane et al, 2007; Li et al, 2009; Malone et al, 2009). In contrast, the surrounding follicle cells (somatic origin) express exclusively Piwi and piRNAs are produced only via the primary pathway (Lau et al, 2009; Li et al, 2009; Malone et al, 2009; Saito et al, 2009). As all three PIWI proteins are expressed in germline cells, accurate systems must be in place to guarantee controlled piRNA biogenesis and PIWI loading. Several recent studies indicate that modular interactions between PIWI proteins and TUDOR domain-containing proteins are part of this control system (Chen et al, 2009; Kirino et al, 2009, 2010; Nishida et al, 2009; Reuter et al, 2009; Vagin et al, 2009). The TUDOR domain is a member of the TUDOR 'royal family', which among others also contains Chromo, plant Agenet, MBT and PWWP domains (Maurer-Stroh et al, 2003). The core TUDOR domain spans ∼60 amino acids and folds into a strongly bent anti-parallel β-sheet with five strands forming a barrel-like fold (Sprangers et al, 2003; Chen et al, 2009; Friberg et al, 2009; Liu et al, 2010a, 2010b). A key function of this domain is to facilitate protein–protein interactions, which often depend on the post-translational methylation of Lysine or Arginine residues in target proteins. Indeed, several methylated Arginine residues have been identified in PIWI-family proteins and at least in some cases specific interactions between PIWI and TUDOR proteins require the symmetric di-methylation of Arginine residues (sDMAs) in PIWI proteins (Kirino et al, 2009, 2010; Nishida et al, 2009; Reuter et al, 2009; Vagin et al, 2009; Huang et al, 2011b). Based on the observed specificity of PIWI–TUDOR interactions, it is possible that an intricate sDMA code allows the controlled recruitment of selected TUDOR domain-containing proteins at specific points of the life cycle of PIWI–piRNA complexes. In Drosophila, six (Tudor, Spindle-E, Krimper, Tejas, Yb and Papi) out of the roughly 20 proteins implicated in the piRNA pathway contain TUDOR domains (Boswell and Mahowald, 1985; Gillespie and Berg, 1995; Lim and Kai, 2007; Malone et al, 2009; Nishida et al, 2009; Olivieri et al, 2010; Patil and Kai, 2010; Qi et al, 2010; Saito et al, 2010; Liu et al, 2011). We therefore decided to systematically analyse all fly TUDOR domain-containing proteins for an involvement in the piRNA pathway. This led to the identification of four novel TUDOR proteins as essential piRNA pathway factors. We characterized in detail the role of CG4771 (Vreteno), a tandem TUDOR domain-containing protein. Vreteno localizes to Yb bodies in follicle cells and to nuage in germline cells and is required for primary piRNA biogenesis in both cell types. Vreteno interacts with the three fly Tdrd12 proteins Yb, CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which have partially overlapping functions in the somatic and germline piRNA pathways. Results Identification and classification of TUDOR domain-containing proteins in Drosophila We mined the Drosophila melanogaster proteome for TUDOR-clan domains (Pfam CL0049) using sensitive sequence–profile (HMMer) and profile–profile comparison methods (Soding et al, 2005). Supplementary Table SI lists all identified proteins and specifies the individual subclasses (see also Figure 1A). For further analysis we focused on the TUDOR-clan domains TUDOR and SMN, which both have been reported to bind sDMA residues (Selenko et al, 2001; Sprangers et al, 2003; Cote and Richard, 2005; Liu et al, 2010a, 2010b). This resulted in 22 proteins containing at least one TUDOR/SMN domain. 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. Download figure Download PowerPoint An alignment of all TUDOR/SMN domains contained in this set indicates three subgroups (Supplementary Figure S1). Groups A (Smn, CG13472 and CG17454) and B (Otu, CG3251) show similarity only to the ∼60 amino-acid TUDOR core. All other sequences cluster together in group C and share significant similarity also N- and C-terminal to the TUDOR core. Characteristic for group C are two 100% conserved amino acids, an Arginine in β4 and an Aspartate in the loop linking β5 and β6 of the extended TUDOR structure (Supplementary Figure S1; marked in green in Figure 1B; Liu et al, 2010a). Based on structural studies, group C sequences represent extended TUDOR domains, which are characterized by a core TUDOR domain tightly interacting with an OB-fold that consists of the N-terminal and C-terminal extensions (Liu et al, 2010a, 2010b). So far, every TUDOR domain-containing protein that has been linked to the piRNA pathway belongs to the extended TUDOR group. To further characterize the set of proteins harbouring extended TUDOR domains, we annotated all additionally contained protein domains and searched for the corresponding mouse orthologues (Figure 1A; Supplementary Figure S2). Most of the fly proteins exhibit strong similarity to their mouse counterparts and the listed pairs in Supplementary Figure S2 are supported by multiple independent orthology assignment methods. CG14303 was linked to Rnf17 based on automated orthology identification (Inparanoid, Compara, OMA) and similarities in the TUDOR domains and an N-terminal B-Box C-terminal domain. We note that the N-terminus of CG14303 is not annotated in FlyBase (lack of EST data), indicating that the similarities between CG14303 and Rnf17 might also include the RING-type zinc finger found in Rnf17. Notably, an N-terminal RING finger could be identified in the Apis and Bombyx CG14303/Rnf17 orthologues. Murine Tdrd12 was assigned to CG11133 and CG31755 based on OrthoMCL (v2:OG2_82474 and v4:OG4_21213). Since both of these proteins share a similar domain composition with the piRNA pathway protein Yb, it appears that Tdrd12 has radiated in Drosophila into three proteins. Indeed, all three fly proteins are more related to each other than to the single mouse or human Tdrd12 proteins. Less obvious was the assignment of Tdrd1, a 4 × TUDOR domain protein with an N-terminal MYND-type zinc finger. Based on domain composition, Tdrd1 might be the single mammalian counterpart of fly CG9925, CG9864 and CG4771, all of which encode besides multiple TUDOR domains also a MYND zinc finger (CG4771 contains in addition an RRM domain). Fly proteins with no assignable mouse counterparts are Krimper as well as the two testes specific proteins CG15042 and CG15930 (the two TUDOR domains of Krimper and CG15042 are highly similar, potentially suggesting a common ancestor). Finally, mouse Tdrd8 seems to lack a detectable fly orthologue. TUDOR/SMN domains often bind peptides with sDMA residues in target proteins. The sDMA-binding pocket resides within the TUDOR core. It consists of four aromatic residues (Figure 1B, marked in red), whose aromatic rings form a cuboid cage and complex the di-methylated guanidine group (Selenko et al, 2001; Sprangers et al, 2003; Cote and Richard, 2005; Liu et al, 2010a, 2010b). An additional conserved Asparagine (Figure 1B, marked in orange) interacts with the sDMA residue via a hydrogen bond (Liu et al, 2010a, 2010b). In sDMA-binding TUDOR domains, the aromatic cage residues are highly conserved and are critical for sDMA binding. We inspected the Drosophila extended TUDOR domains for aromatic cage residues. The alignment in Figure 1B indicates that while a set of TUDOR domains harbours all of these important residues at the exact same position, numerous TUDOR domains seemingly lost the ability to bind sDMA residues due to multiple amino-acid exchanges at critical positions. Nevertheless, many of the identified proteins contain at least one TUDOR domain with an intact aromatic cage and therefore likely interact with sDMA residues. We finally analysed the RNA expression pattern of all TUDOR/SMN genes via the adult Drosophila Fly Atlas (Chintapalli et al, 2007). This showed a strong bias for genes with extended TUDOR domains to be expressed in ovaries and/or testes, further suggesting a link to piRNA biology (Figure 1A; Supplementary Figure S3). Defining the set of TUDOR proteins with critical roles in the ovarian piRNA pathway The implication of several TUDOR proteins in piRNA biology and their often gonad-specific expression prompted us to genetically test all proteins with extended TUDOR domains for their involvement in the piRNA pathway. Defects in the piRNA pathway lead to sterility and to a substantial accumulation of transposon transcripts in ovaries. We therefore assayed these phenotypes in females where individual TUDOR domain-containing proteins were knocked down via RNAi specifically in the ovarian soma (marked in green in Figure 2A and B) or in the germline (marked in beige in Figure 2A and B). 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.). Download figure Download PowerPoint RNAi in the follicular epithelium (soma), where only the primary piRNA pathway is active, was based on tj-GAL4 driven dsRNA-hairpin constructs (hp-lines) from the VDRC (Vienna Drosophila RNAi Centre) library (Dietzl et al, 2007; Olivieri et al, 2010). For the germline we expressed short hairpin constructs (sh-lines) with the germline-specific MTD-GAL4 driver, which allows robust knockdowns (Haley et al, 2008; Ni et al, 2011). In addition, we took advantage of the observation that VDRC hp-lines induce potent RNAi in the germline if expressed in conjunction with Dicer-2 (Sidney Wang and Sarah Elgin, personal communication). Figure 2B illustrates specificity and efficacy of the soma and germline-specific knockdowns using the piRNA biogenesis factor Armitage as an example. Integrity of the somatic piRNA pathway was monitored via a gypsy-lacZ construct that accurately reports piRNA-mediated silencing in follicle cells (Figure 2C; Sarot et al, 2004; Olivieri et al, 2010). Integrity of the germline piRNA pathway was monitored via the steady-state RNA levels of the two transposons HeT-A and blood (Figure 2D). In addition, we determined female fertility rates (percentage of hatched eggs) for all knockdowns (Figure 2E). The two germline knockdown approaches yielded in nearly all cases identical results. We attribute the three exceptions (CG9925-hp, yu-sh, CG14303-sh; Figure 2D and E) to off-target effects or non-functional RNAi lines. Seven TUDOR proteins scored as putative piRNA pathway components (CG4771, CG11133, Tejas, CG14303, Spindle-E, Krimper, Yb). All four factors that had previously been shown to be essential pathway members (Spindle-E, Krimper, Tejas, Yb) were identified. In agreement with the literature, Spindle-E, Krimper and Tejas scored only in the germline knockdowns while Yb scored only in the soma assay (Lim and Kai, 2007; Malone et al, 2009; Szakmary et al, 2009; Olivieri et al, 2010; Patil and Kai, 2010). Papi and Tudor—though previously implicated in the pathway—did not result in transposon de-silencing or sterility. This is in agreement with the literature as both proteins are dispensable for fertility and corresponding mutant ovaries contain no or only slightly elevated transposon RNA levels (Nishida et al, 2009; Liu et al, 2011). We note that the grandchild-less phenotype for Tudor (Boswell and Mahowald, 1985) is recapitulated in the Tudor germline knockdowns. In addition to the known factors, germline knockdowns of three uncharacterized proteins (CG14303, CG4771, CG11133; Figure 2D and E) resulted in sterility and transposon silencing defects. Out of these, CG4771 was also identified as an essential component for the somatic piRNA pathway (Figure 2C) and we therefore decided to characterize this factor in more detail. Vreteno (CG4771) is an essential piRNA pathway factor CG4771 is localized on the third chromosome (Figure 3A) and encodes a protein with two extended TUDOR domains (Figure 3B). The C-terminal TUDOR domain might possess sDMA-binding activity (Figure 1B) and the relevant aromatic cage residues are conserved in distantly related Drosophila species (Figure 3B). In addition, CG4771 harbours an N-terminal RRM domain and a highly conserved zinc finger belonging to the MYND family (C2C4HC). 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. Download figure Download PowerPoint To verify that CG4771 is a piRNA pathway factor, we obtained genetic alleles of this gene. Females homozygous for the P-insertion HP36220 (Bloomington), which is inserted into the 5′UTR of CG4771 (Figure 3A) were sterile and laid eggs that exhibited defects in dorso-ventral patterning as evidenced by a high percentage of fused dorsal appendages. This is a common phenotype of piRNA pathway mutants and stems from the activation of the Chk2 DNA damage pathway, presumably caused by widespread DNA damage originating from uncontrolled transposon activity (Chen et al, 2007; Klattenhoff et al, 2007). However, in HP36220 mutants only the blood element was de-repressed, although germline-specific and soma-specific knockdowns of CG4771 clearly de-repressed also HeT-A and ZAM, respectively (Figure 3C). This suggested that HP36220 is a hypomorphic allele. Indeed, nuclear Piwi localization in the mutant was impaired, yet to a lesser degree than in armitage or Yb mutants (Figure 3D; Olivieri et al, 2010). We therefore generated an additional allele by mobilizing the HP36220 element. Out of 280 analysed excision events, one line (CG4771[Δ1]) exhibited a more pronounced phenotype as homozygous females failed to lay eggs. The ovarian morphology of CG4771[Δ1] mutants strongly resembled those of armitage or zucchini null ovaries (Pane et al, 2007; Olivieri et al, 2010). In some egg chambers, we observed besides the oocyte nucleus a single giant nurse cell nucleus, indicating severe defects in cytokinesis. Based on the morphology of these egg chambers (Figure 3E), we initially named CG4771 'avocado'. While this work was under review, CG4771 was named 'vreteno' in FlyBase by the Lehmann group ('vreteno' means 'spindle' in Bulgarian, referring to the spindle class phenotype of eggs laid by CG4771 mutants) and we therefore adopted this name for consistency reasons. In vreteno[Δ1] homozygous ovaries, germline- and soma-specific transposons were severely de-repressed, indicating the stronger nature of this allele (Figure 3F). This was paralleled by a more pronounced defect in nuclear Piwi accumulation (compare Figure 3G and D). To verify that the vreteno[Δ1] phenotype is due to defects in the CG4771 locus, we restored fertility (not shown), transposon silencing (Figure 3F) and nuclear Piwi localization (Figure 3G) to wild-type levels by introducing a genomic rescue construct that expresses GFP-tagged Vreteno under its endogenous regulatory regions (Figure 3A). As this rescue construct also contained the complete loci for HP1c and CG6985, we measured steady-state RNA levels of all three genes in ovaries of vreteno[Δ1] mutants and of the GFP–vreteno rescued animals (Figure 3H). This confirmed the specificity of the Δ1 allele for the vreteno locus. Immunofluorescence analysis with an antibody recognizing the Vreteno N-terminus further indicated that also the protein is essentially not detectable in ovaries from vreteno[Δ1] mutants (Figure 3I). We also analysed the requirement of vreteno for the piRNA pathway in males. Towards this end, we measured steady-state levels of the transposons mdg1 and copia as well as of the repetitive Stellate locus that is under control of the piRNA pathway in testes. This indicated a requirement of vreteno for copia and Stellate silencing, supported by the observation that silencing was fully restored in males expressing a GFP–vreteno rescue construct (Supplementary Figure S4). Taken together, vreteno encodes a novel piRNA pathway factor that is essential for the ovarian and testes piRNA pathways. Vreteno is required for primary piRNA biogenesis in soma and germline Defects in primary piRNA biogenesis (e.g. in armitage or zucchini mutants) result in a collapse of piRNA populations in follicle cells, in a severe reduction of most germline piRNA species and in defects in Piwi's nuclear accumulation accompanied by a significant loss of Piwi protein (Pane et al, 2007; Malone et al, 2009; Haase et al, 2010; Olivieri et al, 2010; Saito et al, 2010). Delocalization and decreased levels of Piwi were also observed in the vreteno[Δ1] mutant (Figure 3D and G). We therefore analysed the effects of loss of Vreteno on PIWI-family proteins and on piRNA populations in the ovarian soma and germline. Similar to an RNAi-mediated Armitage knockdown, knockdown of Vreteno in follicle cells led to an almost complete loss of Piwi protein in these cells (Figure 4A), indicating defects in pr
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