Aubergine and pi RNA s promote germline stem cell self‐renewal by repressing the proto‐oncogene Cbl
2017; Springer Nature; Volume: 36; Issue: 21 Linguagem: Inglês
10.15252/embj.201797259
ISSN1460-2075
AutoresPatricia Rojas‐Ríos, Aymeric Chartier, Stéphanie Pierson, Martine Simonelig,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle13 October 2017Open Access Source DataTransparent process Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl Patricia Rojas-Ríos Patricia Rojas-Ríos mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Aymeric Chartier Aymeric Chartier mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Stéphanie Pierson Stéphanie Pierson mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Martine Simonelig Corresponding Author Martine Simonelig [email protected] orcid.org/0000-0002-3731-6979 mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Patricia Rojas-Ríos Patricia Rojas-Ríos mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Aymeric Chartier Aymeric Chartier mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Stéphanie Pierson Stéphanie Pierson mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Martine Simonelig Corresponding Author Martine Simonelig [email protected] orcid.org/0000-0002-3731-6979 mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France Search for more papers by this author Author Information Patricia Rojas-Ríos1, Aymeric Chartier1, Stéphanie Pierson1 and Martine Simonelig *,1 1mRNA Regulation and Development, Institute of Human Genetics, UMR9002 CNRS-Université de Montpellier, Montpellier Cedex 5, France *Corresponding author. Tel: +33 4 34 35 99 59; E-mail: [email protected] The EMBO Journal (2017)36:3194-3211https://doi.org/10.15252/embj.201797259 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 Abstract PIWI proteins play essential roles in germ cells and stem cell lineages. In Drosophila, Piwi is required in somatic niche cells and germline stem cells (GSCs) to support GSC self-renewal and differentiation. Whether and how other PIWI proteins are involved in GSC biology remains unknown. Here, we show that Aubergine (Aub), another PIWI protein, is intrinsically required in GSCs for their self-renewal and differentiation. Aub needs to be loaded with piRNAs to control GSC self-renewal and acts through direct mRNA regulation. We identify the Cbl proto-oncogene, a regulator of mammalian hematopoietic stem cells, as a novel GSC differentiation factor. Aub stimulates GSC self-renewal by repressing Cbl mRNA translation and does so in part through recruitment of the CCR4-NOT complex. This study reveals the role of piRNAs and PIWI proteins in controlling stem cell homeostasis via translational repression and highlights piRNAs as major post-transcriptional regulators in key developmental decisions. Synopsis PIWI proteins – such as Aubergine (Aub) – protect the fly germline from aberrant transposon activation but also facilitate self-renewal and differentiation of germline stem cells by repressing translation of specific mRNAs. Aub is intrinsically required for self-renewal and differentiation of Drosophila germline stem cells (GSCs). Aub function in GSC self-renewal requires the protein to be loaded with piRNAs. Aub interacts with CCR4-NOT and represses specific mRNAs to regulate GSC self-renewal. The Cbl proto-oncogene, a regulator of hematopoietic stem cells, is required for GSC homeostasis. Aub and CCR4-NOT repress Cbl mRNA translation to control GSC self-renewal. Introduction The regulation of gene expression at the mRNA level is fundamental for many biological and developmental processes. In recent years, Piwi-interacting RNAs (piRNAs) have emerged as novel key players in the regulation of gene expression at the mRNA level in several models. These 23- to 30-nucleotide-long non-coding RNAs are loaded into specific Argonaute proteins, the PIWI proteins (Ishizu et al, 2012; Guzzardo et al, 2013). Classically, piRNAs repress transposable element expression and transposition in the germline. They are largely produced from transposable element sequences and target transposable element mRNAs by complementarity, which induces their cleavage through the endonuclease activity of PIWI proteins and represses their expression. Recent studies have shown that piRNAs also target protein-coding mRNAs, leading to their repression by PIWI-dependent mRNA cleavage or via the recruitment of the CCR4-NOT deadenylation complex. This regulation is required for embryonic patterning in Drosophila (Rouget et al, 2010; Barckmann et al, 2015), sex determination in Bombyx mori (Kiuchi et al, 2014), and degradation of spermiogenic mRNAs in mouse sperm (Gou et al, 2014; Goh et al, 2015; Watanabe et al, 2015; Zhang et al, 2015). piRNAs involved in the regulation of protein-coding mRNAs in Drosophila embryos are produced in the female germline and provided maternally. An open question is whether this function of the piRNA pathway in post-transcriptional control of gene expression plays a role in the biology of germ cells and germline stem cells (GSCs). In the Drosophila ovary, two to three GSCs are localized in the anterior-most region of each ovariole and self-renew throughout the adult life, giving rise to all germ cells. GSCs in contact with somatic niche cells divide asymmetrically to produce a new stem cell that remains in contact with niche cells (self-renewal) and another cell that differentiates into a cystoblast, upon losing the contact with the niche. Subsequently, the cystoblast undergoes four rounds of synchronous division with incomplete cytokinesis to produce a cyst of 16 interconnected germ cells, of which one cell is specified as the oocyte and the other 15 cells become nurse cells (Fig 1A). Figure 1. Intrinsic role of Aub in GSC self-renewal and differentiation A. Schematic diagram of a germarium showing the somatic cells (blue) and the germline cells (green). The spectrosomes and fusomes are shown in orange. The different regions of the germarium are indicated. Region 1: dividing cysts; region 2: selection of the oocyte; region 3: egg chamber with posteriorly localized oocyte. GSCs, germline stem cells; CB, cystoblast. B–E. Immunostaining of germaria from 7-day-old females with anti-Vasa (green, B–D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) Examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss. F. Quantification of mutant germaria with 0–1 GSC, or with GSC tumors shown in (B–E). The number of scored germaria (n) is indicated on the right graph. Error bars represent standard deviation. ***P-value < 0.001, ns, non-significant, using the χ2 test. G–H'. Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). I. Quantification of germaria containing at least one clonal GSC at 7, 14, and 21 days after clonal induction. 50 to 219 germaria were analyzed per condition. Error bars represent standard deviation. J. Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. Error bars represent standard deviation. ***P-value < 0.001 using the two-tailed Student's t-test. Data information: Scale bars: 10 μm in (B–E) and (G–H'). Download figure Download PowerPoint Two features make Piwi unique with respect to the other two Drosophila PIWI proteins, Aubergine (Aub) and Argonaute 3 (Ago3). First, it represses transposable elements at the transcription level through a nuclear function, whereas Aub and Ago3 act by endonucleolytic cleavage of transposable element mRNAs in the cytoplasm; and second, it plays a role in the somatic and germ cells of the ovary, whereas aub and ago3 function is restricted to germ cells. piwi function in GSC biology has long been addressed. piwi is required in somatic escort cells (which surround GSCs) for GSC differentiation, as well as intrinsically in GSCs for their maintenance and differentiation (Cox et al, 1998, 2000; Jin et al, 2013; Ma et al, 2014). One molecular mechanism underlying Piwi function in GSC biology has recently been proposed to involve its direct interaction with Polycomb-group proteins of the PRC2 complex, leading to indirect massive gene deregulation through reduced PRC2 binding to chromatin (Peng et al, 2016). Regulation of c-Fos by Piwi at the mRNA level in somatic niche cells has also been reported to contribute to the role of Piwi in GSC maintenance and differentiation (Klein et al, 2016). Translational control acting intrinsically in GSCs plays a major role in the switch between self-renewal and differentiation. Two molecular pathways ensure GSC self-renewal through translational repression of differentiation factor mRNAs: the microRNA pathway, and the translational repressors Nanos (Nos) and Pumilio (Pum; Slaidina & Lehmann, 2014). Nos and Pum bind to and repress the translation of mRNAs that encode the differentiation factors Brain tumor (Brat) and Mei-P26, through the recruitment of the CCR4-NOT deadenylation complex (Harris et al, 2011; Joly et al, 2013). In turn, cystoblast differentiation depends on Bag of marbles (Bam), the major differentiation factor forming a complex with Mei-P26, Sex lethal (Sxl), and Benign gonial cell neoplasm (Bgcn) to repress nos mRNA translation; Pum interacts with Brat in these cells to repress the translation of mRNAs encoding self-renewal factors (Li et al, 2009b, 2012, 2013; Harris et al, 2011; Chau et al, 2012). Aub has a distinctive role in protein-coding mRNA regulation. In the early embryo, Aub binds several hundred maternal mRNAs in a piRNA-dependent manner and induces the decay of a large number of them during the maternal-to-zygotic transition (Barckmann et al, 2015). Aub-dependent unstable mRNAs are degraded in the somatic part of the embryo and stabilized in the germ plasm. These mRNAs encode germ cell determinants, indicating an important function of Aub in embryonic patterning and germ cell development. Indeed, Aub recruits the CCR4 deadenylase to nos mRNA and contributes to its deadenylation and translational repression in the somatic part of the embryo. This Aub-dependent repression of nos mRNA is involved in embryonic patterning (Rouget et al, 2010). Here, we address the role of aub in GSC biology. We show that aub is autonomously required in GSCs for their self-renewal. This aub function is independent of bam transcriptional repression in the GSCs and partly independent of activation of the Chk2-dependent DNA damage checkpoint. Aub is also involved in GSC differentiation; aub mutant defect in GSC differentiation is less frequent and involves the Chk2-dependent DNA damage checkpoint. Using an Aub point-mutant form that cannot load piRNAs, we show that piRNAs are required for GSC self-renewal. Genetic and physical interactions indicate that Aub function in GSCs involves interaction with the CCR4-NOT deadenylation complex. Importantly, we identify Casitas B-cell lymphoma (Cbl) mRNA as a target of Aub in GSCs. Cbl acts either as a tumor suppressor or a proto-oncogene depending on its mutations, which lead to myeloid malignancies in humans (Sanada et al, 2009). Cbl encodes an E3 ubiquitin ligase that negatively regulates signal transduction of tyrosine kinases; it plays a role in hematopoietic stem cell homeostasis, maintaining quiescence, and preventing exhaustion of the stem cell pool (An et al, 2015). We show that Aub acts to maintain a low level of Cbl protein in GSCs and that this repression of Cbl mRNA by Aub is essential for GSC self-renewal. Furthermore, we find that Cbl is required for GSC differentiation, thereby identifying a role for Cbl in the regulation of yet another stem cell lineage. This study reveals the function of Aub and piRNAs in GSC self-renewal through the translational repression of Cbl mRNA, thus highlighting the role of the piRNA pathway as a major post-transcriptional regulator of gene expression in key developmental decisions. Results Aub is intrinsically required in GSCs for their self-renewal and differentiation Aub and Ago3 are expressed in GSCs, and we addressed their function in GSC biology (Brennecke et al, 2007; Gunawardane et al, 2007). GSCs can be recognized by their anterior localization in the germarium as well as the presence of the spectrosome, an anteriorly localized spherical organelle in contact with the niche, which is enriched in cytoskeletal proteins (Fig 1A). Cystoblasts also contain a spectrosome that is randomly located in the cell, whereas cells in differentiating cysts are connected by the fusome, a branched structure derived from the spectrosome (Fig 1A). GSCs and differentiating cells were analyzed by immunostaining with an anti-Hts antibody that labels the spectrosome and fusome, and anti-Vasa, a marker of germ cells. We used aubHN2 and aubQC42 strong or null alleles (Schupbach & Wieschaus, 1991) to address the role of Aub in GSC biology. Immunostaining of ovaries with anti-Hts and anti-Vasa revealed strong defects in both GSC self-renewal and differentiation, in aubHN2/QC42 mutant ovaries at 7, 14, and 21 days. A large proportion of aubHN2/QC42 germaria had 0–1 GSC indicating GSC loss, and this defect increased over time (Fig 1B, C and F). A lower proportion of germaria showed differentiation defects, observed as tumors containing undifferentiated cells with spectrosomes (Fig 1D). This phenotype did not markedly increase with time (Fig 1F). Both aubHN2/QC42 phenotypes were almost completely rescued following expression of GFP-Aub with the germline driver nos-Gal4, indicating that both defects were due to aub loss of function in germ cells (Fig 1E and F). Because GSC loss was the most prominent defect in aub mutant ovaries, we focused on this phenotype. We used clonal analysis as an independent evidence to confirm the intrinsic role of aub in GSCs for their self-renewal. Wild-type and aub mutant clonal GSCs were generated using the FLP-mediated FRT recombination system (Golic & Lindquist, 1989) and quantified at three time points after clone induction. We first verified that aub clonal GSCs did not express Aub (Fig EV1A and A'). The percentage of germaria with wild-type clonal GSCs was stable over time (Fig 1G and I). In contrast, the percentage of germaria with aub mutant clonal GSCs strongly decreased with increasing time after clone induction, showing that aub mutant GSCs cannot self-renew (Fig 1H and I). The presence of aub mutant clonal differentiated cysts marked with fusomes indicated that aub mutant GSCs were lost by differentiation (Fig 1H). To confirm this conclusion, we used anti-cleaved Caspase 3 staining to record cell death and address whether the loss of aub mutant GSCs could be due to apoptosis. The number of GSCs expressing cleaved Caspase 3 was low and similar in control (aubHN2/+) and aub mutant GSCs (Fig EV1B–D), indicating that aub mutant GSCs did not undergo cell death. Click here to expand this figure. Figure EV1. GSC loss in aub mutant is independent of apoptosis and of Bam expression A, A'. aubHN2 clonal GSCs do not express Aub protein. Immunostaining of mosaic germaria with anti-GFP (green) and anti-Aub (red) 7 days after clonal induction. DAPI (blue) was used to visualize DNA. The white and yellow arrowheads indicate the control aubHN2/+ and the aubHN2 clonal GSCs, respectively. B–C'. aub mutant GSCs do not die by apoptosis. Immunostaining of control aubHN2/+ (B, B') and mutant aubHN2/QC42 (C, C') germaria with anti-cleaved Caspase3 (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. GSCs are outlined. D. Quantification of germaria shown in (B–C') with cleaved Caspase3-positive GSCs in 7-, 14- and 21-day-old females. The percentages are not statistically different using the χ2 test. E–F'. aub mutant GSCs do not express Bam. Immunostaining of control aubHN2/+ (E, E') and mutant aubHN2/QC42 (F, F') germaria with anti-Bam (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. GSCs are outlined. G. Quantification of mutant germaria with 0–1 GSC shown in Fig 2A and B, showing that the GSC loss remains high in chk2 aub double mutant. The genotypes are indicated at the top. The number of scored germaria (n) is indicated in Fig 2C. Download figure Download PowerPoint Next, we asked whether the GSC self-renewal defect in aub mutant ovaries could result from Bam expression in GSCs. Anti-Bam immunostaining of aub mutant ovaries demonstrated that aub mutant GSCs did not express Bam (100% n = 90, Fig EV1E–F'). Finally, we determined the division rate of aubHN2 GSCs by counting the number of cysts produced by a clonal marked mutant GSC and dividing it by the number of cysts produced by a control unmarked GSC in the same germarium (Jin & Xie, 2007). As expected, the division rate of wild-type GSCs (FRT40A chromosome) was close to 1 (0.95), whereas that of aubHN2 mutant GSCs was 0.67, indicating a slower division rate in aub mutant GSCs (Fig 1J). To address the role of Ago3 in GSC biology, we used ago3 mutant alleles that contain premature stop codons, ago3t1, ago3t2, and ago3t3 (Li et al, 2009a). No GSC loss was recorded in the mutant combination ago3t2/t3 in 7-, 14-, or 21-day-old females, showing that GSC self-renewal was not affected in the ago3 mutant. We checked that Aub expression was similar in wild-type and ago3 mutant GSCs (Fig EV2A–B'). In contrast, ago3t2/t3 females showed a prominent defect in GSC differentiation, with a large proportion of germaria having a higher number of undifferentiated germ cells with a spectrosome (two to four in control germaria, versus six to nine in ago3t2/t3 germaria; Fig EV2C–F). Click here to expand this figure. Figure EV2. GSC tumor phenotype in ago3 mutant germaria A–B'. Immunostaining of wild-type (A, A') and ago3t1/t3 mutant (B, B') germaria with anti-Aub (green) showing that Aub expression is not affected in ago3 mutant GSCs. DAPI (blue) was used to visualize DNA. White and yellow arrowheads indicate wild-type and mutant GSCs, respectively. C, D. Immunostaining of control ago3t2/+ (C) and ago3t2/t3 mutant (D) germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs, and the white arrow indicates the increased number of cells with spectrosomes. E, F. Quantification of germaria shown in (C, D) with increased number of spectrosomes (E) and quantification of spectrosomes per germarium (F). The number of scored germaria (n) is indicated. ***P-value < 0.001 using the χ2 test in (E) and the two-tailed Student's t-test in (F). Data information: Scale bars: 10 μm in (A–D). Download figure Download PowerPoint Together, these results demonstrate that Aub is required intrinsically in GSCs for their self-renewal and differentiation. Aub maintains GSCs by preventing their differentiation independently of Bam expression. In contrast, Ago3 is specifically involved in GSC differentiation. Aub function in GSC self-renewal is partly independent of Chk2 The Chk2-dependent DNA damage checkpoint is activated in several piRNA pathway mutants, leading to developmental defects during mid-oogenesis and, in turn, defective dorsoventral and anteroposterior embryonic patterning (Klattenhoff et al, 2007; Pane et al, 2007). These developmental defects are partially rescued in double mutants of Chk2 kinase (mnk mutant) and different piRNA pathway components. DNA damage in piRNA pathway mutants is thought to result from transposable element transposition. Mobilization of P-elements in crosses that induce hybrid dysgenesis (i.e., the crossing of females devoid of P-elements with males that contain P-elements) leads to a block in GSC differentiation, which is partially rescued by mutation in Chk2 (Rangan et al, 2011). To address whether the defects in GSC self-renewal and differentiation in aub mutant ovaries might be due to activation of the Chk2-dependent checkpoint, we analyzed mnkp6 aubHN2/QC42 double-mutant ovaries using anti-Hts and anti-Vasa immunostaining. The aub mutant tumor phenotype of undifferentiated cell accumulation was almost completely rescued by mnkp6, demonstrating that this phenotype depended on Chk2 (Fig 2A–C). In contrast, the GSC loss phenotype was only partially rescued by mnkp6 and remained visible in a large proportion of double mutant germaria (Figs 2A–C and EV1G), showing that this defect was in part independent of Chk2 checkpoint activation. Figure 2. The role of Aub in GSC self-renewal is partially independent of Chk2 and requires its loading with piRNAs A, B. Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. C. Quantification of the number of GSCs per non-tumorous germarium, and of germaria with GSC tumors, in the indicated genotypes. The number of scored germaria (n) is indicated. Error bars represent standard deviation. The two-tailed Student's t-test and the χ2 test were used in the left and right panels, respectively. ***P-value < 0.001, **P-value < 0.01, *P-value < 0.05. D–F. Immunostaining of germaria from 7-day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). G. Quantification of mutant germaria with 0–1 GSC shown in (D–F). The number of scored germaria (n) is indicated. Error bars represent standard deviation. ***P-value < 0.001, ns, non-significant, using the χ2 test. Data information: Scale bar: 10 μm in (A, B) and (D–F). Download figure Download PowerPoint These results reveal that the mild defect in GSC differentiation in the aub mutant is due to activation of the DNA damage checkpoint. In contrast, the prominent GSC self-renewal defect is partly independent of the DNA damage checkpoint activation, consistent with an additional more direct role of Aub in GSC self-renewal. Aub loading with piRNAs is required for GSC self-renewal To determine whether the role of Aub in GSC self-renewal depends on its loading with piRNAs, we used an Aub double point mutant in the PAZ domain that is unable to bind piRNAs (AubAA; Barckmann et al, 2015). In contrast to UASp-GFP-Aub, which was able to rescue the GSC loss phenotype in aub mutant flies when expressed with nos-Gal4 (Fig 1E and F), expression of UASp-GFP-AubAA at similar levels (Fig EV3A–C) failed to rescue this phenotype (Fig 2D–G). These data indicate that Aub loading with piRNAs is required for GSC self-renewal. To confirm this result, we used a piRNA pathway mutant in which piRNA biogenesis is strongly compromised. In the absence of Ago3, piRNAs are produced through Aub/Aub homotypic ping-pong (Li et al, 2009a) and these piRNAs could be used in Aub-dependent regulation in GSCs, consistent with the lack of GSC self-renewal defect in ago3 mutant. In contrast, armitage (armi) encodes an RNA helicase that is essential for piRNA biogenesis (Cook et al, 2004; Malone et al, 2009). Immunostaining of armi1/72 ovaries with anti-Hts and anti-Vasa revealed a GSC loss that increased with time (Fig EV3D–F), showing a function for armi in GSC self-renewal. Click here to expand this figure. Figure EV3. Expression of GFP-Aub transgenes in GSCs, and GSC loss phenotype in armi mutant A–B'. GFP-Aub and GFP-AubAA are expressed at similar levels in GSCs using the nos-Gal4 driver. Immunostaining of nos-Gal4/UASp-GFP-Aub (A, A') and nos-Gal4/UASp-GFP-AubAA (B, B') ovaries with anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs. C. Quantification of GFP-Aub and GFP-AubAA protein levels in GSCs using fluorescence intensity of immunostaining with anti-GFP shown in (A', B'). Fluorescence intensity was measured in arbitrary units using the ImageJ software. Horizontal bars correspond to the mean and standard deviation. ns, non-significant using the two-tailed Student's t-test. The number of cells analyzed is shown in the figure as dots or squares. D, E. GSC self-renewal defect in armi mutant. Immunostaining of control armi1/+ (D) and armi1/72.1 mutant (E) germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; the white arrow indicates reduced number of GSCs. F. Quantification of germaria with 0–1 GSC shown in (D, E), in 7-, 14- and 21-day-old females. The number of scored germaria (n) is indicated. Error bars represent standard deviation. ***P-value < 0.001 using the χ2 test. Data information: Scale bars: 10 μm in (A–B'), (D) and (E). Download figure Download PowerPoint We conclude that Aub function in GSC self-renewal depends on its loading with piRNAs. Aub interacts with the CCR4-NOT complex for GSC self-renewal Previous reports have shown that PIWI proteins can recruit the CCR4-NOT deadenylation complex to repress mRNAs at the post-transcriptional level (Rouget et al, 2010; Gou et al, 2014). In the Drosophila embryo, Aub is in complex with CCR4, independently of RNA (Rouget et al, 2010). To address whether Aub might act through a similar mode of action in GSCs, we analyzed Aub and CCR4 colocalization. CCR4 is present diffusely in the cytoplasm and accumulates in cytoplasmic foci, in GSCs (Joly et al, 2013). Aub also has a diffuse distribution in the cytoplasm and is present in foci that surround the nucleus collectively referred to as "nuage" (Harris & Macdonald, 2001). Colocalization occurred in diffusely distributed pools of proteins and occasionally in foci (Fig 3A–A''), consistent with deadenylation not taking place in foci (Joly et al, 2013). We then overexpressed CCR4-HA in GSCs using nos-Gal4 and found that CCR4-HA was able to recruit Aub in discrete cytoplasmic regions where CCR4-HA had accumulated, consistent with the presence of CCR4 and Aub in the same complex in GSCs (Fig 3B–B''). Coimmunoprecipitation experiments in ovaries revealed that GFP-Aub was able to coprecipitate the NOT1, NOT3, and CCR4 subunits of the CCR4-NOT deadenylation complex, either in the presence or absence of RNA (Fig 3C). We checked that the coprecipitation of CCR4-NOT subunits was maintained with the mutant form of Aub that does not load piRNAs, GFP-AubAA (Fig 3D). Figure 3. Physical and genetic interaction between Aub and the CCR4-NOT complex A–B''. Immunostaining of wild-type (A–A'') or nos-Gal/UASp-CCR4-HA (B–B'') germaria with anti-Aub (red), and anti-CCR4 or anti-HA (green). GSCs are shown in (A–A''). White arrowheads in (B–B'') indicate cytoplasmic accumulation of CCR4-HA colocalized with Aub in GSCs. C. Co-immunoprecipitation (IP) of NOT1, NOT3, and CCR4 with GFP-Aub in ovaries. Wild-type (WT, mock IP) or nos-Gal4/UASp-GFP-Aub (GFP-Aub) ovarian extracts were immunoprecipitated with anti-GFP, either in the absence or the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, anti-NOT3, and anti-CCR4. Inputs are extracts prior to IP. D. Co-IP of NOT1 and NOT3 with GFP-Aub or GFP-AubAA in ovaries. Wild-type (WT, mock IP), nos-Gal4/UASp-GFP-Aub (GFP-Aub), or nos-Gal4/UASp-GFP-AubAA (GFP-AubAA) ovarian extracts were immunoprecipitated with anti-GFP in the presence of RNase A. Western blots were revealed with anti-GFP, anti-NOT1, and anti-NOT3. Inputs are extracts prior to IP. E–G. Genetic interaction between aub and twin in GSC self-renewal. Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. H. Quantification of mutant germaria with no GSC in 3-, 7-, and 14-day-old females of the genotypes shown in (D–F). The number of scored germaria (n) is indicated. ***P-value < 0.001 using the χ2 test. Data information: Scale bars: 10 μm in (A–B'') and (E–G). Source data are available online for this figure. Source Data for Figure 3C and D [embj201797259-sup-0003-SDataFig3.zip] Download figure Download PowerPoint The twin
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