Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms
2021; Springer Nature; Volume: 22; Issue: 7 Linguagem: Inglês
10.15252/embr.202051342
ISSN1469-3178
AutoresPui Yuen Chung, Keisuke Shoji, Natsuko Izumi, Yukihide Tomari,
Tópico(s)Insect Resistance and Genetics
ResumoArticle11 May 2021free access Transparent process Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms Pui Yuen Chung Pui Yuen Chung orcid.org/0000-0002-4085-6685 Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Keisuke Shoji Keisuke Shoji orcid.org/0000-0003-0183-654X Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Natsuko Izumi Natsuko Izumi orcid.org/0000-0002-0065-9257 Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yukihide Tomari Corresponding Author Yukihide Tomari [email protected] orcid.org/0000-0001-8442-0851 Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Pui Yuen Chung Pui Yuen Chung orcid.org/0000-0002-4085-6685 Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Keisuke Shoji Keisuke Shoji orcid.org/0000-0003-0183-654X Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Natsuko Izumi Natsuko Izumi orcid.org/0000-0002-0065-9257 Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yukihide Tomari Corresponding Author Yukihide Tomari [email protected] orcid.org/0000-0001-8442-0851 Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Author Information Pui Yuen Chung1,2, Keisuke Shoji1, Natsuko Izumi1 and Yukihide Tomari *,1,2 1Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan 2Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan *Corresponding author. Tel: +81 3 5841 7839; E-mail: [email protected] EMBO Reports (2021)22:e51342https://doi.org/10.15252/embr.202051342 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 Figures & Info Abstract PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposable elements and safeguard fertility in germ cells. Many protein factors required for piRNA biogenesis localize to perinuclear ribonucleoprotein (RNP) condensates named nuage, where target silencing and piRNA amplification are thought to occur. In mice, some of the piRNA factors are found in discrete cytoplasmic foci called processing bodies (P-bodies). However, the dynamics and biological significance of such compartmentalization of the piRNA pathway remain unclear. Here, by analyzing the subcellular localization of functional mutants of piRNA factors, we show that piRNA factors are actively compartmentalized into nuage and P-bodies in silkworm cells. Proper demixing of nuage and P-bodies requires target cleavage by the PIWI protein Siwi and ATP hydrolysis by the DEAD-box helicase BmVasa, disruption of which leads to promiscuous overproduction of piRNAs deriving from non-transposable elements. Our study highlights a role of dynamic subcellular compartmentalization in ensuring the fidelity of piRNA biogenesis. Synopsis P-bodies in silkworm cells harbor key piRNA factors. Dynamic exchange of piRNA factors between nuage and P-bodies is impaired by ectopic expression of a Siwi catalytic mutant, which leads to aberrant production of mRNA-derived piRNAs. Silkworm piRNA factors are compartmentalized into nuage and P-bodies. Both Siwi and BmVasa catalytic activities are required for proper nuage/P-body partitioning. Disruption of nuage/P-body partitioning prompts mis-production of mRNA-derived piRNAs. Introduction In animal germ cells, ~ 24–31-nt small RNAs termed PIWI-interacting RNAs (piRNAs) program PIWI proteins and induce silencing of target genes (Ozata et al, 2019). The majority of piRNAs are complementary to transposable elements (TEs) and play a central role in transposon silencing and germ cell development (Siomi et al, 2011; Weick & Miska, 2014; Ozata et al, 2019). While some nuclear PIWIs silence gene expression at the transcriptional level, many PIWI proteins are cytoplasmic and cleave target RNAs with their endonucleolytic (slicer) activity (Siomi et al, 2011; Ozata et al, 2019). These cytoplasmic PIWI proteins are often localized in germline-specific membrane-less organelles termed nuage (Eddy, 1974, 1975; Lim & Kai, 2007; Aravin et al, 2009; Shoji et al, 2009; Patil & Kai, 2010). Studies in model animals including fruit flies, mice, and silkworms suggested that nuage is a site for a feed-forward piRNA amplification pathway called the ping-pong cycle (Brennecke et al, 2007; Gunawardane et al, 2007; Aravin et al, 2008; Kawaoka et al, 2009). This pathway requires at least two PIWI proteins and couples target cleavage to piRNA biogenesis by handing the 3′ cleavage product of a piRNA-loaded PIWI protein to a counterpart PIWI protein (Brennecke et al, 2007; Gunawardane et al, 2007). The handing process, albeit elusive, is thought to be aided by nuage core proteins, including the DEAD-box helicase Vasa and a list of Tudor domain-containing proteins (Lim & Kai, 2007; Malone et al, 2009; Xiol et al, 2014). Besides nuage, the outer membrane of mitochondria is another important site for piRNA biogenesis. Zucchini and Trimmer, the nucleases required for processing long piRNA precursors into mature piRNAs, are localized on the mitochondrial surface (Choi et al, 2006; Saito et al, 2010; Han et al, 2015; Mohn et al, 2015; Izumi et al, 2016, 2020). Nuage is often found cemented between mitochondria within the perinuclear region and is thus also called intermitochondrial cement in mice, and yet they are distinguishable from each other. Therefore, highly organized communication between these cytoplasmic compartments is likely necessary for proper piRNA biogenesis and function (Aravin et al, 2009; Shoji et al, 2009; Huang et al, 2014; Ge et al, 2019; Ishizu et al, 2019). Indeed, the RNA helicase Armitage is known to shuttle between nuage and mitochondria, facilitating stepwise RNA processing within these two compartments in fly ovaries (Ge et al, 2019; Ishizu et al, 2019). Processing bodies (P-bodies) are membrane-less ribonucleoprotein (RNP) condensates which contain factors related to mRNA degradation and microRNA (miRNA) pathways (Decker & Parker, 2012; Luo et al, 2018). In mouse gonocytes, P-bodies are also implicated in the piRNA pathway (Aravin et al, 2009). These gonocytes express two PIWI proteins: MILI and MIWI2. MILI localizes to intermitochondrial cement or pi-bodies, which clump between mitochondria just like nuage in other species (Aravin et al, 2009). The other PIWI protein MIWI2 is accumulated in distinct cytoplasmic condensates, named piP-bodies, together with canonical P-body proteins including the mRNA decapping enzyme DCP1a, ATP-dependent helicase DDX6 and scaffold protein TNRC6 (Aravin et al, 2009). Given that the production of MIWI2-piRNAs requires the target cleavage by MILI-piRNAs (Aravin et al, 2008; Fazio et al, 2011; Manakov et al, 2015), functional crosstalk between pi-bodies and piP-bodies must fuel the ping-pong cycle in mice. However, this functional crosstalk was not found in Drosophila melanogaster, the most well-studied piRNA model, and as a result, how piP-bodies contribute to piRNA biogenesis has remained unclear. Bombyx mori (silkworm) ovary-derived BmN4 is one of the few cell-lines which harbor a fully functional ping-pong cycle (Kawaoka et al, 2009). To date, several nuage piRNA factors have been characterized in BmN4, namely the two PIWI clade proteins Siwi and BmAgo3 (Kawaoka et al, 2009), DEAD-box helicase BmVasa (Xiol et al, 2014) and Tudor domain-containing proteins BmSpnE and BmQin (Nishida et al, 2015). A previous study has reported that wild-type BmVasa immunoprecipitates exclude the BmSpnE/BmQin heterodimer and vice versa, despite that both interact with Siwi (Nishida et al, 2015). This observation suggested that, in BmN4, Siwi is actively partitioned into the two distinct complexes. However, the mechanism and significance of this partitioning have remained unclear. Here, by using live cell imaging, we comprehensively characterized the localization of silkworm piRNA factors and defined "piP-bodies" in BmN4 cells, where BmVasa is excluded but the BmSpnE/BmQin heterodimer is enriched together with the canonical P-body protein BmDcp2. Unlike wild-type Siwi that normally localizes in nuage, slicer-deficient Siwi was excluded from nuage and mislocalized in piP-bodies. Conversely, depletion of BmVasa caused aberrant aggregation of Siwi with piP-body components, suggesting that BmVasa is a core regulator of nuage/piP-body partitioning. Importantly, we found that disruption of the nuage/piP-body partitioning leads to promiscuous overproduction of piRNAs deriving from non-TE, protein-coding mRNAs. We propose that silkworm germ cells ensure the fidelity of the piRNA biogenesis pathway by actively enforcing its subcellular compartmentalization. Results Silkworm piRNA factors are compartmentalized into nuage and piP-bodies To dissect the compartmentalization of piRNA biogenesis machineries, we adopted live cell imaging of BmN4 cells. Although CRISPR/Cas9-mediated knockout was successfully achieved on a few genes in BmN4 cells (Zhu et al, 2015; Izumi et al, 2016), our preliminary results suggest that long-term deletion of Siwi causes cell lethality. Moreover, precise genome editing including epitope tagging is still technically challenging in BmN4 cells. We therefore employed the strategy to express proteins of interest with an N-terminal fluorescent tag (AcGFP or mCherry) in naive BmN4 cells, in the presence of endogenous wild-type counterparts. Given that the slicer activity of PIWI proteins is the driving force of the ping-pong cycle, we first examined the subcellular localization of the slicer-defective mutants of Siwi (D670A) and BmAgo3 (D697A) in BmN4 cells. We found that similar to wild-type BmAgo3 and wild-type Siwi, BmAgo3-D697A slicer mutant remains colocalized with BmVasa in nuage. In contrast, Siwi-D670A was largely dislodged from nuage and instead localized in distinct cytoplasmic condensates (Fig 1A and B). Figure 1. Cytoplasmic compartmentalization of the silkworm piRNA pathway A, B. Colocalization of AcGFP-Siwi, AcGFP-BmAgo3, and mCherry-BmVasa in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant dissociates from BmVasa foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm. C, D. Colocalization of AcGFP-Siwi, AcGFP-BmAgo3, and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm. E. Heatmap of colocalization ratio between fluorescence protein-tagged piRNA factors. Each colocalization ratio is quantified by using ImageJ plugin Comdet (see Materials and Methods) and averaged from n = 3 independent z-stacks. Darker color represents more frequent colocalization. Known nuage proteins (BmVasa, BmAgo3, Siwi) form a cluster at the top-left corner. Siwi-D670A mutant, but not BmAgo3-D697A, clusters with BmSpnE, BmQin, and BmDcp2 at the bottom-right corner, representing piP-bodies. See also Figs EV1 and EV2. F. Schematic diagram showing the proposed spatial compartmentalization of the BmN4 piRNA pathway. Nuage components: BmVasa, BmAgo3, Siwi; piP-body components: BmSpnE, BmQin, and BmDcp2. Siwi-D670A mutation leads to the accumulation of the mutant itself in piP-bodies and colocalization with BmSpnE and BmQin. See also Fig EV1. Download figure Download PowerPoint We next asked whether Siwi-D670A may have localized to P-bodies, inspired by the study of mammalian piP-bodies (Aravin et al, 2009). We used the well-conserved decapping enzyme BmDcp2 as a P-body marker in silkworm cells (Franks & Lykke-Andersen, 2008; Zhu et al, 2013). Remarkably, Siwi-D670A foci completely overlapped with BmDcp2 foci, whereas wild-type Siwi as well as both the wild-type and D697A mutant of BmAgo3 did not (Fig 1C and D). Siwi-D670A also colocalized with BmSpnE and BmQin (Fig EV1A). In fact, even in the absence of Siwi-D670A, BmSpnE, and BmQin, but not BmVasa, were observed in the same foci as BmDcp2 (Fig EV1B), which is reminiscent of the mouse piP-bodies (Aravin et al, 2009) and in line with a previous biochemical study on the silkworm piRNA pathway (Nishida et al, 2015). Click here to expand this figure. Figure EV1. Colocalization of Siwi-D670A, BmSpnE, BmQin in P-bodies Colocalization of Siwi-D670A with BmSpnE or BmQin in non-perinuclear cytoplasmic condensates. Scale bar 8 μm. Colocalization of wild-type piRNA factors with BmDcp2 in P-bodies. BmSpnE and BmQin, but not BmVasa, are localized in BmDcp2-containing P-bodies. Scale bar 8 μm. Localization of Siwi piRNA loading mutant (Y607E) in BmN4 cells. (Top) Siwi-Y607E is largely dispersed in the cytoplasm. (Bottom) Introduction of Y607E mutation into Siwi-D670A compromised its colocalization with BmDcp2. Scale bar 8 μm. Colocalization of endogenous BmMael with BmDcp2, Siwi, and Siwi-D670A. BmMael is partially localized to the peripheral region of BmDcp2-containing P-bodies and is strongly colocalized with both wild-type and mutant Siwi in nuage and P-bodies, respectively. Scale bar 10 μm (cell), 2 μm (foci, enlarged region from the white box). Colocalization of stably expressed GFP-BmArmi with Siwi and Siwi-D670A. BmArmi is partially localized to both Siwi (nuage) and Siwi-D670A (P-bodies). Scale bar 10 μm (cell), 2 μm (foci, enlarged region from the white box). Download figure Download PowerPoint To validate the presence of piRNA factors in silkworm P-bodies in a more comprehensive manner, we performed a cross-colocalization assay with 8 factors: Siwi, Siwi-D670A, BmAgo3, BmAgo3-D697A, BmVasa, BmSpnE, BmQin, and BmDcp2. Particle–particle colocalization ratios were quantified and plotted as a heat map (Fig 1E). As expected, a strong colocalization cluster consisting of core nuage proteins (BmVasa, BmAgo3, and Siwi) was observed (top-left corner in Fig 1E). In contrast, BmSpnE, BmQin, and BmDcp2 formed a cluster distinct from those nuage proteins (bottom-right corner in Fig 1E), representing P-bodies. Thus, a subset of piRNA factors is present exclusively in P-bodies of silkworm cells (Fig 1F), as in mouse gonocytes but unlike in fruit flies. Importantly, our comprehensive colocalization assay confirmed that Siwi-D670A, but not BmAgo3-D697A, resides in P-bodies, unlike wild-type Siwi in nuage. This shift in localization requires piRNA loading, as unloaded Siwi (Y607E, 5′ binding pocket mutant. Kawaoka et al, 2011) was largely dispersed in the cytoplasm (Fig EV1C top) and the introduction of Y607E mutation into Siwi-D670A abolished its P-body localization (Fig EV1C bottom). Maelstrom (Mael) is an essential factor for the assembly of piP-bodies in mice (Aravin et al, 2009). In silkworm BmN4 cells, BmMael partially colocalizes with BmDcp2 in piP-bodies but also with wild-type Siwi in nuage (Fig EV1D). Interestingly, when the D670A slicer mutant of Siwi was expressed, BmMael exhibited extensive colocalization with Siwi-D670A in P-bodies. Similarly, BmArmi, which partially colocalizes with wild-type Siwi in nuage and with a mitochondrial marker (Patil et al, 2017; Izumi et al, 2020), was found colocalized with Siwi-D670A in P-bodies (Fig EV1E). These data suggest a possibility that BmMael and BmArmi, which transiently enter multiple subcellular compartments in normal BmN4 cells, are trapped in P-bodies in Siwi-D670A-expressing cells. To eliminate the possibility of overexpression artifacts, we performed Western blotting and confirmed that our expression system yields proteins at levels comparable to their endogenous counterparts (Fig EV2A). Furthermore, by replacing the constitutive promoter OpIE2 (pIZ) with an inducible promoter Tet-On (pTet), we successfully reduced the expression level of the epitope-tagged protein down to detection limits (Fig EV2A and B). Since the Western blotting results do not reflect the transfection efficiency and the protein concentration at the single cell level, we further analyzed pIZ- and pTet-AcGFP-Siwi-transfected cells with flow cytometry (Fig EV2C). The results suggested that the pIZ construct yielded around 2.47-folds more proteins than the endogenous Siwi, while the pTet construct yielded at most 0.08-folds (> 30-folds decrease in the GFP signal). Importantly, all key combinations of colocalization hold true for the pTet-transfected cells (Fig EV2D). We therefore concluded that inactivation of the slicer activity of Siwi renders it to leave nuage and instead joins BmSpnE/BmQin-containing P-bodies in silkworm cells (Fig 1F). We herein refer to these piRNA factors-containing P-bodies as piP-bodies. Click here to expand this figure. Figure EV2. Reduction in protein expression level with Tet-On inducible promoter Western blotting of endogenous counterparts and the epitope-tagged version of the protein-of-interest (POI), either expressed by an OpIE2 promoter (pIZ) or a Tet-On system (pTet). Most of the pIZ constructs express epitope-tagged POI (upper bands) at levels comparable to the endogenous counterparts (lower bands) and all of the pTet constructs express POI at significantly lower levels than the pIZ constructs and the endogenous counterparts. Relative whole-cell fluorescence intensity of AcGFP-tagged POI expressed by an OpIE2 promoter (pIZ) or a Tet-On system (pTet). Flow cytometry analysis of pIZ-AcGFP-Siwi or pTet-AcGFP-Siwi transfected cells. Naive BmN4 was used as a negative control (0.18% GFP-positive cells (GFPpos); See Appendix Figure S1). pIZ construct yielded 8.45% GFPpos, while pTet yielded 2.5% GFPpos that were roughly 30–100 folds dimmer than pIZ-GFPpos. FSC: Forward scatter. For the gating strategy, see Appendix Figure S1. Colocalizations of Siwi-D670A, BmSpnE, and BmQin with BmDcp2, or BmVasa-E339Q with Siwi-D670A were not affected by the reducing POI expression level with pTet. Scale bar 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Validation of dsRNA-mediated knockdown of piRNA factors, effects of BmMael knockdown, and BmVasa ATPase mutation Western blotting of AcGFP-Siwi, wild-type (WT) or D670A, and the co-expressed mCherry-BmQin or mCherry-BmSpnE. Expression of Siwi-D670A did not affect the expression level of BmQin or BmSpnE. Validation of dsRNA-mediated knockdown of epitope-tagged piRNA factors by Western blotting. Validation of dsRNA-mediated knockdown of endogenous piRNA factors by Western blotting. Depletion of BmMael results in partial segregation of Siwi-D670A foci from BmDcp2 foci. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmMael (dsBmMael, n = 24 cells) reduced colocalization ratio between Siwi and BmDcp2, compared with control (dsRLuc, n = 24 cells). Representative data from N = 3 independent experiments are shown. P-value was calculated by asymptotic Wilcoxon rank sum test. Centre line, median; box limits, lower (Q1), and upper (Q3) quartiles; whiskers, 1.5 × interquartile range (IQR); points, outliers. Colocalization of BmAgo3 and BmQin with BmVasa-E339Q (ATPase mutant). Scale bar 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Definition of potential-TE genes and analysis of piRNA hallmarks MA plot of differential piRNA expression analysis between two naive small RNA libraries. Reads longer than 25-nt are mapped to predicted genes (Silkbase GeneModel) that do not have a BLAST (tblastx) hit with known transposons (Silkbase Transposon Database). Mean piRNA expression with an RPM higher than 24 is defined as potential TE-derived piRNAs (potential TEs). Length distribution of piRNA reads mapped to different groups of predicted genes. Peak length of piRNAs mapped to TEs and potential TEs are both 27-nt, while peak length of piRNAs mapped to non-TEs is 28-nt. Note that despite that Siwi-D670A causes significant upregulation of 28-nt piRNA from non-TEs, the expression of siRNA/miRNA at 20-nt peak remains unchanged between Siwi and Siwi-D670A libraries. Nucleotide bias of the 1st and 10th nucleotide of non-TE-derived piRNAs in Siwi and Siwi-D670A libraries. (Top) Ratios of uracil as the 1st nucleotide of piRNAs (1 U bias) in TEs and potential TEs are roughly 80% in both libraries. In non-TEs, 1 U bias is retained at 38% in Siwi-WT library and 45% in Siwi-D670A library, which is higher than the control (the frequency of uracil in all CDSs, 23%, black dotted line), suggesting the mapped reads contain bona fide piRNAs. (Bottom) A bias of favoring adenine at the 10th nucleotide (10A bias) can be found in ping-pong piRNAs. 10th nucleotides of TE piRNAs (~ 44%) and potential TE piRNAs (~ 38%) are mildly biased to adenine, compared with the control frequency of adenine in all CDSs (29%, black dotted line). Non-TE-derived piRNAs do not have an apparent 10A bias (~ 30%, similar to the control), suggesting that non-TE-derived piRNAs are generated independently of the ping-pong cycle. Analysis of the strand directionality of mapped piRNA reads. Strand directionality is calculated by counting and dividing sense reads over antisense reads, and the mean strand directionality is calculated between the 2 naive libraries. A strand directionality of 1.0 means that only sense reads are mapped to the predicted CDS region, while −1.0 means only antisense reads are mapped. In non-TEs genes, most of the mapped piRNAs are in sense direction. In contrast, potential TEs genes are mapped with piRNA reads from both directions, suggesting the involvement of ping-pong machineries. Dashed line: expression threshold value (24 RPM) for the definition of potential TE genes. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Upregulated non-TE piRNAs in Siwi-D670A expressed cells are 2′-O-methylated MA plots of differential piRNA expression analysis of Siwi/Siwi-D670A- overexpressed (OE) cells against mCherry-OE cells. Reads longer than 25-nt are mapped to predicted genes (Silkbase GeneModel) and non-TE genes derived reads are upregulated only in Siwi-D670A-OE cells. NaIO4 treatment did not deplete the upregulation. Split violin plots of piRNA expression fold change between mCherry (control) and Siwi (WT/D670A) overexpressed libraries with or without NaIO4 treatment. Non-TE piRNAs (red, 628 genes) upregulated or TE piRNAs (blue, 811 genes) remained unchanged in Siwi-D670A-OE cells have a similar distribution and mean value regardless of NaIO4 treatment. Bonferroni-corrected P-values and the effect sizes (r) were calculated by asymptotic Wilcoxon rank sum test. Box plot: Centre line, median; box limits, lower (Q1), and upper (Q3) quartiles; whiskers, 1.5 × interquartile range (IQR); points, outliers. MA plot of NaIO4 + against NaIO4 – small RNA libraries from Siwi-D670A-OE cells. A mild depletion of the less abundant piRNAs is observed similarly with TE, potential TE and non-TE piRNAs. Length distribution of mCherry-, Siwi-, and Siwi-D670A-OE cells (± NaIO4 treatment). Two peaks (20-nt and 27-nt) correspond to siRNA/miRNA and piRNA respectively. The first peak (20-nt) is depleted after NaIO4 treatment, leading to an increase of the read counts of the piRNA peak (27-nt) in those libraries. Download figure Download PowerPoint Siwi slicer mutant forms solid-like aggregates with BmSpnE and BmQin in piP-bodies Previous biochemical studies revealed that slicer-defective Siwi-D670A can load piRNA but fails to cleave target RNAs (Matsumoto et al, 2016). We therefore reasoned that Siwi-D670A is likely to stick on target RNAs as a "frozen" pre-cleavage complex. Supporting this idea, fluorescence recovery after photobleaching (FRAP) experiments showed that Siwi-D670A has a drastically reduced molecule exchange rate compared with wild-type Siwi (Fig 2A). Moreover, 5% 1,6-Hexanediol, aliphatic alcohol used to distinguish liquid-like condensates and solid-like aggregates (Kroschwald et al, 2015, 2017), dissolved wild-type Siwi foci but not Siwi-D670A foci (Fig 2B). Figure 2. Siwi-D670A specifically accumulates with BmSpnE and BmQin at piP-bodies Fluorescence Recovery After Photobleaching (FRAP) experiment on AcGFP-Siwi and AcGFP-Siwi-D670A. Red dotted lines represent recovery traces of Siwi-D670A foci (n = 36), and black dotted lines represent recovery traces of Siwi foci (n = 36). Solid lines represent locally estimated scatterplot smoothing (LOESS) curve, and the gray areas represent 95% confidence level interval. Fluorescence intensity was quantified with the softWoRx software and normalized to minimum and maximum in a 0-1 scaling. (Left) Effect of 1,6-Hexanediol treatment on AcGFP-Siwi and AcGFP-Siwi-D670A foci. Z-stacks were taken prior to the addition of 1,6-Hexanediol or medium and after 30 min incubation at RT. Z-projections of the middle 6 μm stacks were then normalized and pseudo-colored with Fire LUT to visualize the Siwi foci. Scale bar 10 μm. Scale bars represent pixel intensity in arbitrary units (A.U.) (Right) High granule/cell intensity ratio of Siwi-D670A persists despite 1,6-Hexanediol treatment. Average intensity ratio from each of the independent Z-stacks (n = 30 per set) was quantified (see Materials and Methods) and depicted as box plots. Representative data from N = 3 independent experiments are shown. P-values were calculated by asymptotic Wilcoxon rank sum test. Points represent outliers. Co-expression of Siwi-D670A increases granule-to-cell intensity ratio of BmSpnE and BmQin foci. (Upper) Representative Z-projections (maximum intensity) of mCherry-BmSpnE and mCherry-BmQin foci. AcGFP signal from FITC channel is not shown. Scale bar 8 μm. (Bottom) Average intensity ratio from each of the independent Z-stacks (n = 6 per set) was quantified (see Materials and Methods) and depicted as box plots. Representative data from N ≥ 3 independent experiments are shown. P-values were calculated by asymptotic Wilcoxon rank sum test. Depletion of BmSpnE or BmQin results in segregation of Siwi-D670A from BmDcp2-containing P-bodies. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmQin (n = 32 cells) or BmSpnE (n = 32 cells) reduces colocalization ratio between Siwi-D670A and BmDcp2, compared with control (dsRLuc, n = 32 cells). Representative data from N = 3 independent experiments are shown. Bonferroni-corrected P-values were calculated by asymptotic Wilcoxon rank-sum test. Points represent data points. See also Fig EV3. Data information: (B-D) Box plots: Centre line, median; box limits, lower (Q1), and upper (Q3) quartiles; whiskers, 1.5 × interquartile range (IQR). Download figure Download PowerPoint Given that Siwi-D670A colocalizes with piRNA factors in piP-bodies, its solid-like aggregates may trap other piP-body factors such as BmSpnE and BmQin. Indeed, during our cross-colocalization assay, we noticed that the presence of Siwi-D670A is coupled to the observation of more focused and brighter foci of BmSpnE and BmQin. By quantitative imaging, we confirmed that the granule-to-whole-cell intensity ratio of these piP-bodies factors was significantly increased by the co-expression of Siwi-D670A compared with that of wild-type Siwi (Fig 2C), while the expression levels of BmSpnE or BmQin were unchanged (Fig EV3A). This suggests that slicer-deficient Siwi-D670A forms solid-like aggregates while trapping BmSpnE and BmQin in piP-bodies. On the other hand, colocalization between Siwi-D670A and the P-body marker BmDcp2 was severely compromised by the depletion of BmQin or BmSpnE (Figs 2D, and EV3B and C). The reduction in the colocalization ratio between Siwi-D670A and BmDcp2 was also observed with BmMael depletion (Fig EV3C and D), despite its partial piP-body localization (Fig EV1D). Thus, recruitment of Siwi-D670A to piP-bodies depends on BmQin, BmSpnE, and BmMael, while the presence of Siwi-D670A renders BmQin and BmSpnE to be anchored in piP-bodies. Siwi shuttles between nuage and P-bodies by changing its interactors Previous immunoprecipitation assays have detected the interaction of wild-type Siwi with BmSpnE and BmQi
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