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• W3106592092 abstract "Article24 November 2020Open Access Source DataTransparent process Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the Caenorhabditis elegans embryo Maria Placentino Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany Search for more papers by this author António Miguel de Jesus Domingues orcid.org/0000-0002-1803-1863 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Jan Schreier orcid.org/0000-0002-5036-2419 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany Search for more papers by this author Sabrina Dietz International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany Quantitative Proteomics Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Svenja Hellmann Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Bruno FM de Albuquerque orcid.org/0000-0001-8483-6822 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Graduate Program in Areas of Basic and Applied Biology, University of Porto, Porto, Portugal Search for more papers by this author Falk Butter orcid.org/0000-0002-7197-7279 Quantitative Proteomics Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author René F Ketting Corresponding Author [email protected] orcid.org/0000-0001-6161-5621 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Institute of Developmental Biology and Neurobiology, Johannses Gutenberg University, Mainz, Germany Search for more papers by this author Maria Placentino Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany Search for more papers by this author António Miguel de Jesus Domingues orcid.org/0000-0002-1803-1863 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Jan Schreier orcid.org/0000-0002-5036-2419 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany Search for more papers by this author Sabrina Dietz International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany Quantitative Proteomics Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Svenja Hellmann Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Bruno FM de Albuquerque orcid.org/0000-0001-8483-6822 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Graduate Program in Areas of Basic and Applied Biology, University of Porto, Porto, Portugal Search for more papers by this author Falk Butter orcid.org/0000-0002-7197-7279 Quantitative Proteomics Group, Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author René F Ketting Corresponding Author [email protected] orcid.org/0000-0001-6161-5621 Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany Institute of Developmental Biology and Neurobiology, Johannses Gutenberg University, Mainz, Germany Search for more papers by this author Author Information Maria Placentino1,2, António Miguel Jesus Domingues1, Jan Schreier1,2, Sabrina Dietz2,3, Svenja Hellmann1, Bruno FM Albuquerque1,4, Falk Butter3 and René F Ketting *,1,5 1Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany 2International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany 3Quantitative Proteomics Group, Institute of Molecular Biology (IMB), Mainz, Germany 4Graduate Program in Areas of Basic and Applied Biology, University of Porto, Porto, Portugal 5Institute of Developmental Biology and Neurobiology, Johannses Gutenberg University, Mainz, Germany *Corresponding author. Tel: +49 6131 3921470; E-mail: [email protected] EMBO J (2021)40:e105280https://doi.org/10.15252/embj.2020105280 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 In Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This silencing can become PRG-1-independent and heritable over many generations, a state termed RNA-induced epigenetic gene silencing (RNAe). How and when RNAe is established, and how it is maintained, is not known. We show that maternally provided 21U RNAs can be sufficient for triggering RNAe in embryos. Additionally, we identify PID-2, a protein containing intrinsically disordered regions (IDRs), as a factor required for establishing and maintaining RNAe. PID-2 interacts with two newly identified and partially redundant eTudor domain-containing proteins, PID-4 and PID-5. PID-5 has an additional domain related to the X-prolyl aminopeptidase APP-1, and binds APP-1, implicating potential N-terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect size and appearance of RNA inheritance-linked Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in C. elegans small RNA silencing. SYNOPSIS Caenorhabditis elegans maternal piRNAs are sufficient to establish and maintain stable epigenetic silencing without further piRNA production (termed RNAe). Here, PID-2 and its newly-found interactors PID-4 and PID-5, are shown to be required for this heritable silencing, in addition to affecting Z granule homeostasis. Maternal piRNAs are sufficient to trigger long-term multigenerational silencing in embryos. Intrinsically-disordered protein PID-2 is required for effective piRNA-induced silencing, and interacts with Tudor-domain proteins PID-4 and PID-5. PID-2, PID-4 and PID-5 are required for efficient inheritance of silencing and maintenance of germline function/germline immortality across generations. PID-2, PID-4 and PID-5 localize to perinuclear foci and modulate size and appearance of RNA inheritance-linked Z granules, balancing 22G RNA populations. Introduction Germ cells are responsible for transmitting genetic information to the next generation. Therefore, genome stability should be tightly controlled in these cells. The integrity of the genome is constantly threatened not only by external factors, such as irradiation and mutagenic agents, but also by intrinsic factors resident in the genome, such as transposable elements (TEs). Consequently, organisms have evolved a variety of mechanisms to counteract these threats. Among these, small RNA pathways often play important roles in controlling TE activity. In many animals, TEs are recognized and silenced in the germline by a specific small RNA pathway: the Piwi pathway. Piwi proteins represent a specific subclade of Argonaute proteins that exert their silencing function upon loading with their cognate small RNA, named piRNA (Piwi-interacting RNA), that specifies the target transcript. The Piwi/piRNA pathway is essential in most organisms for TE silencing, but also TE-unrelated effects have been well-described (Ghildiyal & Zamore, 2009; Malone & Hannon, 2009; Ketting, 2011; Siomi et al, 2011; Ozata et al, 2019). The main and likely only active Piwi protein of Caenorhabditis elegans is PRG-1; it binds to piRNAs, which in C. elegans are named 21U RNAs, to form a silencing complex. In contrast to other organisms, loss of the PRG-1/21U RNA pathway in C. elegans causes the reactivation of only a limited set of transposable elements, for instance Tc3 (Das et al, 2008), and does not cause immediate sterility (Cox et al, 1998; Batista et al, 2008; Das et al, 2008; Wang & Reinke, 2008), even though germ cells are progressively lost over generations (mortal germline phenotype, Mrt) (Simon et al, 2014). The discrepancy between the Piwi-mutant phenotypes observed in C. elegans and other animals can be explained by the fact that PRG-1 initiates a silencing response that is executed by a different set of Argonaute proteins—the worm-specific Argonaute proteins (WAGOs)—while in other studied model systems this does not happen. Upon target recognition by PRG-1, an RNA-dependent RNA polymerase (RdRP) is recruited to the targeted transcript, which is used as a template for the synthesis of complementary small RNAs, named 22G RNAs. For this step, the RdRP RRF-1 is required, as well as so-called Mutator proteins (Zhang et al, 2011; Phillips et al, 2012; Phillips et al, 2014). The 22G RNAs, characterized by the 5ʹ triphosphate group resulting from the RdRP-driven synthesis, are loaded onto WAGO proteins, such as HRDE-1 and WAGO-1 (Gu et al, 2009; Ashe et al, 2012; Buckley et al, 2012; Shirayama et al, 2012), that reinforce the silencing started by PRG-1. Occasionally, in a seemingly stochastic and poorly understood manner, this silencing can become independent of PRG-1 itself and self-sustainable. This form of silencing is extremely stable and can be maintained across many generations in the absence of PRG-1. It is characterized by the deposition of heterochromatic marks at the targeted locus, depends on HRDE-1 and Mutator activity, and it is known as RNAe (RNA-induced epigenetic gene silencing) (Ashe et al, 2012; Luteijn et al, 2012; Shirayama et al, 2012). RNAe can thus explain why transposons remain silenced in the absence of PRG-1. Indeed, in prg-1;hrde-1 double mutants, lacking both 21U RNAs and RNAe, the activity of Tc1 transposons increases to levels comparable to Mutator mutants, indicating that HRDE-1 activity is sufficient to maintain Tc1 silencing in prg-1 mutants (de Albuquerque et al, 2015). PRG-1/21U RNA complexes can recognize a target transcript via imperfect base-pair complementarity, allowing up to four mismatches (Bagijn et al, 2012; Lee et al, 2012). As a consequence of this mismatch tolerance, PRG-1 is potentially able to recognize and silence many different sequences, including endogenous genes (Bagijn et al, 2012; Gu et al, 2012). Another small RNA pathway, guided by 22G RNAs bound to the Argonaute protein CSR-1, has been implicated in counteracting such PRG-1-mediated silencing of genes that should be expressed (Claycomb et al, 2009; Gu et al, 2009; Lee et al, 2012; Shirayama et al, 2012; Conine et al, 2013; Seth et al, 2013; Wedeles et al, 2013; Shen et al, 2018). CSR-1-bound 22G RNAs are made by the RdRP EGO-1 in a mostly Mutator-independent manner (Claycomb et al, 2009; Gu et al, 2009). Interestingly, an opposite scenario has also been described: PRG-1 has been shown to direct Mutator activity to non-CSR-1 targets in embryos that set up a 22G RNA silencing response de novo (de Albuquerque et al, 2015; Phillips et al, 2015). These seemingly contradictory findings—CSR-1 counteracting inappropriate PRG-1 targeting versus PRG-1 directing Mutator activity away from CSR-1 targets—may be explained by considering that two different developmental stages have been analysed to arrive at the proposed models. The protective role of CSR-1 has been seen in the adult germline (Claycomb et al, 2009; Gu et al, 2009; Lee et al, 2012; Shirayama et al, 2012; Conine et al, 2013; Seth et al, 2013; Wedeles et al, 2013; Shen et al, 2018), whereas the protective role of PRG-1 likely operates in embryos (de Albuquerque et al, 2015; Phillips et al, 2015). Possibly, PRG-1 has different modes of actions at these two developmental stages. Another result that indicates differential PRG-1 activities in adults versus embryos comes from studies on HENN-1, the enzyme that 2ʹ-O-methylates 21U RNAs. In adults, 21U RNA levels are not affected by loss of HENN-1 (Kamminga et al, 2012), while in embryos 21U RNAs are reduced in henn-1 mutants (Billi et al, 2012; Montgomery et al, 2012). Given that 2ʹ-O-methylation has been shown to stabilize small RNAs, in particular when they base pair extensively to their targets (Ameres et al, 2010), it is feasible that PRG-1 recognizes targets with near-perfect complementarity to its cognate 21U RNA only in the embryo and employs more relaxed 21U RNA targeting in the adult germline. Indeed, maternally provided PRG-1 protein is required to establish PRG-1-driven silencing of a 21U RNA sensor transgene that has perfect 21U RNA homology, suggesting that this silencing is set up during early development, and not in the adult germline (de Albuquerque et al, 2014). Whether the maternal contribution of PRG-1 is sufficient to induce silencing has not been tested thus far. A third small RNA pathway is driven by so-called 26G RNAs (Yigit et al, 2006; Han et al, 2009; Conine et al, 2010; Billi et al, 2014). These are made by the RdRP enzyme RRF-3, which acts in a large protein complex containing well-conserved proteins such as Dicer, GTSF-1 and ERI-1 (Kennedy et al, 2004; Duchaine et al, 2006; Thivierge et al, 2012; Billi et al, 2014; Almeida et al, 2018). These 26G RNAs can be bound by the Argonaute protein ERGO-1, or by two closely related paralogs, the Argonaute proteins ALG-3 and ALG-4 (ALG-3/-4). ERGO-1 mostly targets transcripts in the female germline and the early embryo, and is required to load the somatic, nuclear Argonaute protein NRDE-3 with 22G RNAs (Han et al, 2009; Gent et al, 2010; Vasale et al, 2010; Billi et al, 2014; Almeida et al, 2019a). The 26G RNAs bound by ERGO-1 require HENN-1-mediated 2ʹ-O-methylation in both the adult germline and the embryo (Billi et al, 2012; Montgomery et al, 2012; Kamminga et al, 2012). ALG-3/-4-bound 26G RNAs are not modified by HENN-1 (Billi et al, 2012; Montgomery et al, 2012; Kamminga et al, 2012) and are specifically expressed in the male gonad (Han et al, 2009; Conine et al, 2010; Conine et al, 2013). Many of the above-mentioned proteins are found in phase-separated structures, often referred to as granules or foci. Mutator proteins that make 22G RNAs are found in so-called Mutator foci, whose formations is driven by MUT-16, a protein with many intrinsically disordered regions (IDRs) (Phillips et al, 2012; Uebel et al, 2018). The RdRP EGO-1, as well as the Argonaute proteins CSR-1, PRG-1 and a number of others, are found in P granules (Batista et al, 2008; Wang & Reinke, 2008; Claycomb et al, 2009; Updike & Strome, 2010), characterized by IDR proteins such as PGL-1 (Kawasaki et al, 1998) and DEPS-1 (Spike et al, 2008), which are also required for P granule formation. Finally, Z granules are marked by the conserved helicase ZNFX-1 and the Argonaute protein WAGO-4 (Ishidate et al, 2018; Wan et al, 2018). Z granules are related to the inheritance of small RNA-driven responses via the oocyte (Ishidate et al, 2018; Wan et al, 2018) and are typically found adjacent to P granules. However, in primordial blastomeres, Z and P granules appear to be merged (Wan et al, 2018). For Z granules, no IDR protein that may drive their formation has been identified yet. The function of ZNFX-1 is also not clear, but it has been demonstrated that it interacts with the RdRP EGO-1 and that it is required to maintain the production of 22G RNAs from the complete length of the targeted transcript (Ishidate et al, 2018). In the absence of ZNFX-1, relatively more 22G RNAs are found to originate from the 5ʹ part of the RdRP substrate, suggesting that ZNFX-1 may have a role in maintaining or relocating the RdRP activity to the 3ʹ end of the substrate. Despite the fact that material exchange between these three types of structures (P, Z granules and Mutator foci) seems obvious, how this may happen is currently unknown. Here, we describe the characterization of a novel gene, pid-2, which we identified from our published “piRNA-induced silencing defective” (Pid) screen (de Albuquerque et al, 2014). Our analyses show that the IDR protein PID-2 is essential for initiation of silencing by maternally provided PRG-1 activity. However, PID-2 is also required for efficient maintenance of RNAe and shows defects in many different small RNA populations indicating that PID-2 does not only act together with PRG-1. Interestingly, we noticed a drop of 22G RNA coverage specifically at the 5ʹ end of RRF-1 substrates, suggesting that PID-2 may be involved in stimulating RdRP activity or processivity. At the subcellular level, PID-2 is found in granules right next to P granules, and the absence of PID-2 affects size and number of Z granules, suggesting that PID-2 itself may also be in Z granules. We also identify two PID-2-interacting proteins, PID-4 and PID-5, with an extended Tudor (eTudor) domain. In addition, PID-5 has a domain that closely resembles the catalytic domain of the X-prolyl aminopeptidase protein APP-1. Loss of both PID-4 and PID-5 phenocopies pid-2 mutants in many aspects, including the effects on small RNA populations and on Z granules. At steady state, both PID-4 and PID-5 are themselves mostly detected close to or within P granules. We hypothesize that the here identified PID-2/-4/-5 proteins have a role in controlling RdRP activity, and do so by affecting protein and/or RNA exchange between different germ granules. Results PID-2 is an IDR-containing protein required for 21U RNA-driven silencing We have previously performed and published a forward mutagenesis screen in which we identified several mutants that are defective for 21U RNA-driven silencing (piRNA-induced silencing defective: Pid) (de Albuquerque et al, 2014). In this screen, the de-silencing of a fluorescent 21U RNA target was used as read-out. The silencing of the transgene depended on both PRG-1 and 22G RNAs (Bagijn et al, 2012), allowing for the isolation of mutants that affect 21U or 22G RNAs; we will refer to this PRG-1-dependent state as 21U sensor(+). Here, we focused our attention on a mutant, defined by the allele xf23, resulting in a point mutation (tgg → tga) that causes a premature stop codon (W122X) in the gene Y48G1C.1. This gene encodes a protein with disordered N- and C-terminal regions (Fig 1A). The rest of the encoded protein is more structured (Fig 1A), even though no predicted domains were detected. We also obtained a publicly available deletion allele of Y48G1C.1, tm1614 (Fig 1A; Barstead et al, 2012). Imaging revealed that animals homozygous for xf23 or tm1614 showed a strongly penetrant silencing defect of the 21U sensor(+), even though the defect is less severe compared with Mutator mutants (Fig 1B). Quantification of the de-silencing induced by both alleles using RT–qPCR revealed 10–20% activation of the 21U sensor(+) compared with Mutator mutants (Fig EV1A). A single-copy transgene expressing 3xFLAG-tagged Y48G1C.1, and to a lesser extent GFP-tagged Y48G1C.1, driven by its endogenous promoter and 3ʹ UTR could rescue the 21U sensor(+) phenotype (Fig EV1B–E). We conclude that the mutation in Y48G1C.1 plays a role in 21U sensor(+) silencing, and named the gene pid-2. Figure 1. PID-2 is a novel factor required for establishing de novo silencing mediated by maternally provided small RNAs Schematic representation of the Y48G1C.1/ pid-2 gene and its mutant alleles (xf23 and tm1614). The line plot displays the predicted disorder of the Y48G1C.1/PID-2 protein, as obtained from PONDR, using the algorithms VL3 and VL-XT. Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor (RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm. Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild-type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented. See Materials and Methods for details. Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm. Crossing scheme to address whether maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm. Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy and depicted in a pie chart. White arrowheads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm. Source data are available online for this figure. Source Data for Figure 1 [embj2020105280-sup-0006-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PID-2 affects 21U sensor expression RT-qPCR of the 21U sensor in the indicated mutant backgrounds. *: strains that received the 21U sensor from a mut-7 background (active). §: strains that received a silenced 21U sensor from a prg-1 background (RNAe). Expression of pmp-3 was used to normalize the data. Error bars reflect the standard deviation, calculated from three technical replicates. We note that the pid-2 strain, that received the 21U sensor originally in an RNAe state, has RNA levels of the 21U sensor comparable to a de-silenced sensor in a pid-2 mutant background. Indeed, upon re-examination of this strain under the microscope, we detected expression of the 21U sensor, indicating that in this isolate RNAe had been lost (also see Fig EV1F). Bars represent the fold change of the 21U sensor expression compared with the control (pmp-3). Error bars represent mean ± SD. For each strain, three replicates have been used. Scheme of the single-copy MosSCI and miniMos transgenes expressing tagged PID-2. The MosSCI transgenes (xfSi144, xfSi145, xfSi146) are driven by the endogenous promoter and 3ʹ UTR and were inserted into a known germline-expressing site on chromosome II, ttTi5605. The miniMos transgenes (xfSi83, xfSi98) instead are driven by a germline-specific promoter (pgl-3) and 3’UTR (tbb-2) and have been randomly inserted in the genome. The xfSi83 transgene was mapped to chromosome II, within the last intron of mpz-1, whereas the transgene xfSi98 was mapped to chromosome V, within the fourth intron of the Y32B12B.4 gene. Crossing strategy to assess the rescue of the pid-2 mutation by the indicated pid-2 transgenes. Quantification of the expression of the 21U sensor (% of animals analysed) in the indicated mutant backgrounds as a measure of the rescue of the pid-2 mutation by the different pid-2 transgenes. Expression was quantified by scoring individuals (ON/OFF) using microscopy. Representative images of the 21U sensor expression (left: mCherry signal; right: DIC) in the indicated genetic backgrounds. For xfSi145, both ON and OFF states are depicted. Gonads are outlined by a dashed line. Scale bar: 25 μm. Quantification of the reactivation of the 21U sensor (RNAe) (indicated as % of plates containing animals with detectable expression), at either 20°C or at 25°C, in the indicated mutant backgrounds. From each of the indicated mutant backgrounds, 20 L2-L3 larvae were singled out and scored to ensure that the 21U sensor was still silenced (RNAe). Then, 10 plates were kept at 20°C and 10 plates were kept at 25°C and chunked regularly to avoid starvation. After 14 days, plates were scored by microscopy for reactivation of the 21U sensor (RNAe). When expression was detected on a plate, the majority of animals showed expression (~60–80%). pid-2(tm1614)1 and pid-2(tm1614)2 represent two independently generated strains with the same genotype (RFK530 and RFK586, respectively). 22G RNA profile on the 21U sensor schematically represented at the bottom, in the indicated mutant backgrounds. The top two panels refer to control strains, whereas the bottom two panels show profiles from strains isolated from an individual in which the 21U sensor was exposed to maternal 21U RNAs only. Both silenced and non-silenced strains were sequenced. In each plot, the average of three biological replicates is represented. The shading represents the standard deviation among the replicates. Representative images of the 21U sensor expression (left: mCherry signal; right: DIC) in the indicated genetic backgrounds. The two top panels represent strains that have been exposed to maternal 21U RNAs only. In the strain depicted on top, the sensor became silenced, whereas in the strain depicted below it did not get silenced. Upon introduction of hrde-1 mutation in the strain carrying a 21U sensor silenced upon exposure to maternal 21U RNAs only, the 21U sensor is reactivated, as shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm. Examples of pid-1;pid-2 double mutant animals, isolated from a growing pid-1;pid-2 double mutant population, showing feminization (upper panel) and pseudo-males (lower panel). The arrow indicates arrayed appearance of oocytes in the feminized animal (upper panel), while it indicates a male-like tail in the pseudo-male (lower panel). The latter also shows characteristics of hermaphrodites (two gonad arms, eggs in uterus and vulva). Scale bar: 100 μm. Source data are available online for this figure. Download figure Download PowerPoint The 21U sensor can also be in a state of RNAe: 21U sensor (RNAe). In this state, its silencing no longer depends on PRG-1, but does still rely on 22G RNAs (Ashe et al, 2012; Luteijn et al, 2012; Shirayama et al, 2012). In contrast to the sensor(+) reactivation experiment, most pid-2 mutant animals did not reactivate the 21U sensor (RNAe) (Fig 1B). Nonetheless, we did detect reactivation of the 21U sensor (RNAe) in some animals, most notably in pid-2(xf23) mutants (Fig 1B). Continuous culturing of independent cultures confirmed recurrent loss of RNAe status in pid-2(xf23) mutants, particularly at elevated temperature (Fig EV1F). Such loss of RNAe was much less frequent in pid-2(tm1614) animals (Fig EV1F). Given that the reactivation of the sensor(+) was also less effective in pid-2(tm1614) mutants (Fig 1B), we assume that pid-2(tm1614) is a weaker allele than pid-2(xf23), and as such only has a very weak phenotype in the more stringent sensor (RNAe) assay, while it has an easily scored phenotype in the sensor(+) assay. RT–qPCR showed that pid-2-mediated reactivation of the 21U sensor (RNAe) transgene resulted in RNA expression levels that were very similar to that of 21U sensor(+) in a pid-2 mutant background (Fig EV1A). We conclude that loss of PID-2 leads to the stochastic loss of the RNAe status of the 21U sensor, implying a role for PID-2 in the inheritance of silencing. PID-2 acts together with HRDE-1 to silence Tc1 transposition Next, we tested whether pid-2(xf23) impaired silencing of the DNA transposon Tc1. We found that pid-2(xf23) animals displayed activa" @default.
• W3106592092 created "2020-12-07" @default.
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• W3106592092 title "Intrinsically disordered protein PID‐2 modulates Z granules and is required for heritable piRNA‐induced silencing in the<i>Caenorhabditis elegans</i>embryo" @default.
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