Paraspeckles are constructed as block copolymer micelles
2021; Springer Nature; Volume: 40; Issue: 12 Linguagem: Inglês
10.15252/embj.2020107270
ISSN1460-2075
AutoresTomohiro Yamazaki, Tetsuya Yamamoto, Hyura Yoshino, Sylvie Souquère, Shinichi Nakagawa, Gérard Pierron, Tetsuro Hirose,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle22 April 2021free access Source DataTransparent process Paraspeckles are constructed as block copolymer micelles Tomohiro Yamazaki Corresponding Author Tomohiro Yamazaki [email protected] orcid.org/0000-0003-0866-5173 Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Search for more papers by this author Tetsuya Yamamoto Tetsuya Yamamoto orcid.org/0000-0002-6786-8299 Institute for Chemical Reaction Design and Discovery, Hokkaido University, Sapporo, Japan Search for more papers by this author Hyura Yoshino Hyura Yoshino Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Sylvie Souquere Sylvie Souquere orcid.org/0000-0002-7768-5293 UMS 3655, AMMICA, Gustave Roussy, Villejuif, France Search for more papers by this author Shinichi Nakagawa Shinichi Nakagawa orcid.org/0000-0002-6806-7493 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Gerard Pierron Gerard Pierron orcid.org/0000-0002-5910-3122 Centre National de la Recherche Scientifique, UMR-9196, Gustave Roussy, Villejuif, France Search for more papers by this author Tetsuro Hirose Corresponding Author Tetsuro Hirose [email protected] orcid.org/0000-0003-1068-5464 Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Tomohiro Yamazaki Corresponding Author Tomohiro Yamazaki [email protected] orcid.org/0000-0003-0866-5173 Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Search for more papers by this author Tetsuya Yamamoto Tetsuya Yamamoto orcid.org/0000-0002-6786-8299 Institute for Chemical Reaction Design and Discovery, Hokkaido University, Sapporo, Japan Search for more papers by this author Hyura Yoshino Hyura Yoshino Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Sylvie Souquere Sylvie Souquere orcid.org/0000-0002-7768-5293 UMS 3655, AMMICA, Gustave Roussy, Villejuif, France Search for more papers by this author Shinichi Nakagawa Shinichi Nakagawa orcid.org/0000-0002-6806-7493 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Gerard Pierron Gerard Pierron orcid.org/0000-0002-5910-3122 Centre National de la Recherche Scientifique, UMR-9196, Gustave Roussy, Villejuif, France Search for more papers by this author Tetsuro Hirose Corresponding Author Tetsuro Hirose [email protected] orcid.org/0000-0003-1068-5464 Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Search for more papers by this author Author Information Tomohiro Yamazaki *,1, Tetsuya Yamamoto2, Hyura Yoshino3, Sylvie Souquere4, Shinichi Nakagawa5, Gerard Pierron6 and Tetsuro Hirose *,1,3 1Graduate School of Frontier Biosciences, Osaka University, Suita, Japan 2Institute for Chemical Reaction Design and Discovery, Hokkaido University, Sapporo, Japan 3Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan 4UMS 3655, AMMICA, Gustave Roussy, Villejuif, France 5Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 6Centre National de la Recherche Scientifique, UMR-9196, Gustave Roussy, Villejuif, France *Corresponding author. Tel: +81 6 6879 4675, E-mail: [email protected] *Corresponding author. Tel: +81 6 6879 4674, E-mail: [email protected] The EMBO Journal (2021)40:e107270https://doi.org/10.15252/embj.2020107270 PDFDownload PDF of article text and main figures.AM PDF 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 Paraspeckles are constructed by NEAT1_2 architectural long noncoding RNAs. Their characteristic cylindrical shapes, with highly ordered internal organization, distinguish them from typical liquid–liquid phase-separated condensates. We experimentally and theoretically investigated how the shape and organization of paraspeckles are determined. We identified the NEAT1_2 RNA domains responsible for shell localization of the NEAT1_2 ends, which determine the characteristic internal organization. Using the soft matter physics, we then applied a theoretical framework to understand the principles that determine NEAT1_2 organization as well as shape, number, and size of paraspeckles. By treating paraspeckles as amphipathic block copolymer micelles, we could explain and predict the experimentally observed behaviors of paraspeckles upon NEAT1_2 domain deletions or transcriptional modulation. Thus, we propose that paraspeckles are block copolymer micelles assembled through a type of microphase separation, micellization. This work provides an experiment-based theoretical framework for the concept that ribonucleoprotein complexes (RNPs) can act as block copolymers to form RNA-scaffolding biomolecular condensates with optimal sizes and structures in cells. SYNOPSIS Paraspeckles scaffolded by NEAT1_2 lncRNAs have characteristic shapes and internal structures distinct from typical LLPS droplets. Using soft matter physics theory, this study reveals that they are built as block copolymer micelles, providing a concept that RNPs act as block copolymers. NEAT1_2 RNPs behave as block copolymer and form paraspeckles as micelles. Length of the NEAT1_2 5′ and 3′ terminal domains and transcription rate of NEAT1_2 determine the internal organization, size, number, and shape of the paraspeckle. NEAT1_2 lacking both the 5′ and 3′ terminal domains form large spherical paraspeckles via macroscopic phase separation. RNPs have the potential to form various structures as block copolymer micelles. Introduction Membraneless organelles, also known as cellular bodies or biomolecular condensates, have attracted much attention because of their involvement in biological processes and pathological conditions, and because of the physical process of their formation by phase separation (Hyman et al, 2014; Banani et al, 2017; Shin & Brangwynne, 2017; Alberti & Dormann, 2019). In particular, liquid–liquid phase separation (LLPS), a type of phase separation, is widely used in a variety of biological processes (Banani et al, 2017; Shin & Brangwynne, 2017; Alberti et al, 2019; Strom & Brangwynne, 2019; Sabari et al, 2020). Many biomolecular condensates contain proteins, such as intrinsically disordered proteins and oligomer-forming proteins, and RNAs (Sawyer et al, 2019; Choi et al, 2020; Sabari et al, 2020). Although the mechanisms of protein phase separation have been extensively studied, the role of RNA remains poorly understood. A class of RNA, termed architectural RNA (arcRNA), plays an essential scaffolding role in the formation of phase-separated condensates in various eukaryotic species from yeast to human (Chujo et al, 2016; Chujo et al, 2017; Yamazaki et al, 2018; Yamazaki et al, 2019; Roden & Gladfelter, 2021). The arcRNAs include several categories of RNA transcripts, mainly long noncoding RNAs (lncRNAs), which are pervasively transcribed from eukaryotic genomes and are important cellular regulatory factors (Quinn & Chang, 2016; Schmitt & Chang, 2017). Among the lncRNAs, non-repetitive lncRNAs, repeat-derived lncRNAs, short tandem repeat-enriched RNAs, and disease-associated repetitive RNAs have been identified as arcRNAs (Sasaki et al, 2009; Chujo et al, 2016; Chujo et al, 2017; Hall et al, 2017; Dumbović et al, 2018; Fox et al, 2018; Yap et al, 2018; Swinnen et al, 2019; Ninomiya & Hirose, 2020). NEAT1_2 lncRNA is a representative arcRNA and constructs paraspeckle nuclear bodies (Chen & Carmichael, 2009; Clemson et al, 2009; Sasaki et al, 2009; Sunwoo et al, 2009). NEAT1_2 lncRNA (22.7 kb) is a longer isoform of the NEAT1 gene and is essential for paraspeckle formation, whereas NEAT1_1 (3.7 kb), a shorter isoform, is not essential for paraspeckle formation (Naganuma et al, 2012). NEAT1_2 lncRNAs are upregulated by several factors, including proteasome inhibition, p53 activation, and viral infections, and play critical roles in various physiological and pathological conditions (Hirose et al, 2014; Imamura et al, 2014; Nakagawa et al, 2014; Standaert et al, 2014; Mello et al, 2017; Nakagawa et al, 2018). At the molecular level in HeLa cells, a single spherical paraspeckle has been shown to contain approximately 50 NEAT1_2 molecules (Chujo et al, 2017). More than 60 paraspeckle proteins (PSPs) are enriched in paraspeckles, and several of these PSPs are required for the paraspeckle formation processes (Naganuma et al, 2012; Kawaguchi et al, 2015; Yamazaki & Hirose, 2015). Proteins, including SFPQ and NONO, are required for the expression of NEAT1_2 lncRNA, and several proteins, including NONO, FUS, and RBM14, are required for paraspeckle assembly (Naganuma et al, 2012; Hennig et al, 2015; Yamazaki et al, 2018). Specifically, oligomerization of NONO through NOPS and coiled-coil domains and interactions through the low-complexity domains of FUS and RBM14 are required for paraspeckle assembly (Hennig et al, 2015; Yamazaki et al, 2018). We have used CRISPR/Cas9-mediated dissection of NEAT1_2 in human haploid HAP1 cells to demonstrate that NEAT1_2 has modular functional RNA domains for RNA stabilization, isoform switching from NEAT1_1 to NEAT1_2, and paraspeckle assembly (Yamazaki et al, 2018). The middle domain of NEAT1_2 contains multiple binding sites for NONO and SFPQ that are necessary and sufficient for paraspeckle assembly through phase separation (Yamazaki et al, 2018). Previous studies using electron microscopy (EM) and super-resolution microscopy (SRM) have shown that paraspeckles can have a spherical or cylindrical shape (Souquere et al, 2010; West et al, 2016). The short axis (Sx) of paraspeckles is constrained (~ 360 nm), while the long axis (Lx) elongates upon transcriptional NEAT1_2 upregulation in cylindrical paraspeckles (Souquere et al, 2010; Yamazaki et al, 2018). Additionally, NEAT1_2 is looped and highly spatially organized within paraspeckles with the 5′ and 3′ ends of NEAT1_2 localized in the shell of the paraspeckle and the middle domain localized in the core of the paraspeckle (Souquere et al, 2010; West et al, 2016; Yamazaki et al, 2018). The 5′ and 3′ domains appear to be bundled and form distinct domains in the shells of the paraspeckles (West et al, 2016). PSPs also show specific patterns of localization within paraspeckles, suggesting a core-shell structure for the paraspeckles (West et al, 2016). These characteristic shapes and the internal organization of paraspeckles are distinct from those of the typical condensates formed by LLPS that are spherical and have non-ordered internal structures, although phase-separated condensates with core-shell or multi-layered architectures have been also reported (e.g., Feric et al, 2016; Fei et al, 2017; Harmon et al, 2018; Boeynaems et al, 2019). At present, it is unknown how the NEAT1_2 lncRNA determines both the paraspeckle shape and the highly ordered NEAT1_2 organization within it. Here, we addressed these questions both experimentally and theoretically. We first experimentally identified the 5′ and 3′ RNA domains of NEAT1_2 that determine the shell localization by dissecting NEAT1_2 lncRNA in vivo. We then applied soft matter physics theories to understand the principles of the formation of the paraspeckle structure. We treated the paraspeckles as amphipathic block copolymer micelles, which form spherical and cylindrical shapes with ordered internal structures analogous to paraspeckles. Our theoretical model could explain and predict the observed behaviors of the paraspeckles formed by wild type (WT) and mutant NEAT1_2 in terms of the internal organization, size, number, and shape. Thus, this study provides a conceptual framework for the formation and structure of nuclear biomolecular condensates with RNA scaffolds. Results The 3′ terminal domain of NEAT1_2 is required for localization of the 3′ end in the shell of paraspeckles In HAP1 WT cells, quantitative analyses using SRM have shown that the 5′ and 3′ ends of NEAT1_2 are localized in the shell of paraspeckles, as previously indicated by EM analyses (Souquere et al, 2010) (Figs 1A and F, and EV1A and B). It has been suggested that the 5′ and 3′ terminal domains of NEAT1_2 determine the shell localization of the 5′ and 3′ ends of the NEAT1_2 transcripts. We found that the truncated 3′ end of the NEAT1_2 Δ16.6–22.6 kb mutant (Δ3′ mutant) was localized in the core of paraspeckles, even in the presence of the 3′-terminal triple-helix structure (Yamazaki et al, 2018). We have also observed that the expression of NEAT1_1 and NEAT1_2 in Δ3′ mutant cells was comparable to that in WT cells (Yamazaki et al, 2018). Then, we further characterized this Δ3′ mutant. In Δ3′ mutant cells, in which no paraspeckle assembly defects were detected (Fig EV1C), quantitative analysis using SRM showed the localization of the 3′ end of the Δ3′ mutant to the core of the paraspeckles, whereas the 5′ end remained localized in the shell (Figs 1A, B, and F, and EV1B). The EM analysis confirmed that the truncated 3′ end of the Δ3′ mutant was localized in the core without affecting the localization of the 5′ end in the shell (Figs 1C and F, and EV1D). 5′ and D2 probes for EM were used to detect the localization in the shells and cores, respectively, in WT cells, as previously reported (Souquere et al, 2010) (Fig EV1E and F). Thus, the 16.6–22.6 kb region of NEAT1_2 is required for the shell localization of the 3′ end of NEAT1_2, but its deletion does not appreciably affect the assembly of the paraspeckles. Figure 1. Deletion of the 3′ terminal domain of NEAT1_2 causes core localization of the 3′ ends of NEAT1_2 within the paraspeckle The schematics of human NEAT1_2 (WT) and the mutants with deletions in the 3′ terminal regions. The NEAT1 transcripts are shown above with a scale. The gray dashed lines represent the deleted regions. The positions of NEAT1 probes used in SRM (blue) and EM (orange) are shown. (left) SRM images of paraspeckles in HAP1 NEAT1 Δ3′ mutant cells (Δ3′) detected by NEAT1_5′ (green) and NEAT1_15k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends in the core or in the core and shell (n = 44). (upper) EM observation of the paraspeckles in Δ3′ cells using NEAT1_D2 probes in the presence of MG132 (5 μM for 17 h). Scale bar, 100 nm. (lower, left) Graph showing the proportion of localization of the NEAT1 region detected by NEAT1_D2 probe (248 gold particles) within the paraspeckles in Δ3′ cells. (lower, right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in Δ3′ cells (n = 17). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). SRM images of the paraspeckles in Δ16.6–20.2 and Δ20.2–22.6 kb cells detected by NEAT1_5′ (green) and NEAT1_3′ or 19k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the core in Δ16.6–20.2 and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h). (Δ16.6–20.2 kb: n = 230, Δ20.2–22.6 kb: n = 99) Schematics of the NEAT1_2 configuration in WT and deletion mutants. Source data are available online for this figure. Source Data for Figure 1 [embj2020107270-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Characterization of NEAT1 mutants with deletions in the NEAT1_2 3′ terminal regions (left) SRM images of the paraspeckles in HAP1 WT cells detected by NEAT1_5′ (green) and NEAT1_3′ (magenta) FISH probes with MG132 treatment (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the shell in WT cells (n = 167). Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in WT, Δ3′, Δ16.6–20.2 kb, and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h; WT: n = 115, Δ3′: n = 21, Δ16.6–20.2 kb: n = 161, and Δ20.2–22.6 kb: n = 89). (left) Detection of NEAT1_2 by single-molecule FISH (smFISH; magenta) in HAP1 WT, Δ3′, Δ16.6–20.2 kb, and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h). Nuclei were stained with DAPI. Scale bar, 10 nm. (right) Quantitation of area and sum intensity per paraspeckle in each cell line (WT: n = 407, Δ3′: n = 314, Δ16.6–20.2 kb: n = 302, and Δ20.2–22.6 kb: n = 609). (***P = 0.0001, ****P < 0.0001, compared with WT: Kruskal–Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). (left) EM observations of the paraspeckles in MG132-treated (5 μM for 17 h) HAP1 Δ3′ cells using NEAT1_5′ probe. Scale bar, 100 nm. (middle) Graph showing the proportion of the localization of NEAT1_5′ probes (838 gold particles) within the paraspeckles in Δ3′ cells. (right) Graph showing the proportion of localization of NEAT1_5′ probes in each paraspeckle in Δ3′ cells (n = 21). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). (left) EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) HAP1 WT cells using NEAT1_5′ probe. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (601 gold particles) within the paraspeckles in WT cells. (right) Graph showing the proportion of localization of NEAT1_5′ probes in each paraspeckle in WT cells (n = 16). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). (left) EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) HAP1 WT cells using the NEAT1_D2 probe. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_D2 probes (461 gold particles) within the paraspeckles in WT cells. (right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in WT cells (n = 24). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). Quantitation of the relative expression levels of NEAT1_1 and NEAT1_2 by RT–qPCR in HAP1 WT, Δ16.6–20.2 kb, and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h). Data are represented as mean ± SD (n = 3). SRM images of the paraspeckles in MG132-treated (5 μM for 6 h) WT cells detected by NEAT1_5′ (green) and NEAT1_19k (magenta) FISH probes. Scale bar, 500 nm. Graph showing the proportion of paraspeckles with localization of the NEAT1_19k probes to the core and shell or the core in WT cells treated with MG132 (5 μM for 6 h; n = 103). Source data are available online for this figure. Download figure Download PowerPoint To determine the precise region of the NEAT1_2 domain that is required for the shell localization, we established two HAP1 mutant cell lines (Δ16.6–20.2 and Δ20.2–22.6 kb; Fig 1A). In both cell lines, the NEAT1_2 expression levels were comparable to that of the WT, and the paraspeckles were formed similarly to the WT (Fig EV1C and G). As shown by SRM, in the majority of the Δ16.6–20.2 kb cells, the 3′ ends of this NEAT1_2 mutant were distributed in both the core and shell of the paraspeckles (Fig 1D and E). In contrast, the 3′ ends of NEAT1_2 in the Δ20.2–22.6 kb cells were mostly detected in the shell, similar to the WT paraspeckles, indicating that this region played only a minor role in the shell localization (Figs 1D and E, and EV1H, and I). Altogether, these data revealed that, unlike the WT and Δ3′ mutant, the Δ16.6–20.2 kb mutant showed a random distribution of the 3′ end within the paraspeckles (Fig 1F); thus, the NEAT1_2 16.6–20.2 kb region has a major role in the proper shell localization of the 3′ end of NEAT1_2. The 5′ terminal domain of NEAT1_2 is required for shell localization of the 5′ end within the paraspeckles We next investigated whether the 5′ terminal domain of NEAT1_2 has a role in the shell localization of the 5′ end. We used two mutant cell lines, the previously established NEAT1 Δ0–0.8 kb and a newly established Δ0–1.9 kb (Δ5′ mutant), in which NEAT1_2 was expressed comparably to that in WT cells, although a NEAT1 0–1 kb deletion has been previously shown to prevent NEAT1_2 accumulation (Yamazaki et al, 2018) (Figs 2A and EV2A). No paraspeckle assembly defects were observed in these cell lines (Fig EV2B). Our SRM analysis showed that the signals of the 5′ end were randomly localized within the paraspeckles for both mutants, whereas the signals of the 3′ end were detected in the shells in both cell lines (Figs 2B and C, and EV2C). Consistent with these data, EM observations confirmed the random localization of the 5′ region of NEAT1_2 in the Δ5′ mutant cells (Fig 2D). We also examined the localization of the middle region of NEAT1_2 in detail using the D2 probe for EM (Fig 2A). The middle region was mainly detected in the inner and middle layers of the paraspeckles in WT cells, as has been previously reported (Souquere et al, 2010), but in the Δ5′ mutant cells, the middle region was mainly detected in the outer and middle layers of the paraspeckles, suggesting that this region had a tendency toward being localized in the shells of the paraspeckles (Fig EV2D). Collectively, these data showed that the 5′ terminal domain of NEAT1 is required for the shell localization of the 5′ end and influences the internal distribution of the NEAT1_2 transcripts within the paraspeckles (Fig 2E). Figure 2. Deletion of 5′ terminal domain of NEAT1_2 causes random distribution of the NEAT1_2 5′ ends within the paraspeckle The schematics of WT NEAT1_2 and mutants with deletions in the 5′ terminal regions are shown in Fig 2A. The positions of NEAT1 probes used in SRM (blue) and EM (orange) are shown. SRM images of the paraspeckles in Δ0–0.8 and Δ0–1.9 kb (Δ5′) cells treated with MG132 (5 μM for 6 h) detected by NEAT1_5′, 1k, and 2k (green) and NEAT1_3′ (magenta) FISH probes. Scale bar, 500 nm. Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in WT, Δ0–0.8 kb, and Δ5′ cells treated with MG132 (5 μM for 6 h). (WT: n = 159, Δ0–0.8 kb: n = 54, Δ5′: n = 48) (left) EM observation of the paraspeckles in Δ5′ cells treated with MG132 (5 μM for 17 h) using NEAT1_5′ probes. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (347 gold particles) within the paraspeckles in Δ5′ cells. (right) Graph showing the proportion of the localization of NEAT1_5′ probes in each paraspeckle in Δ5′ cells (n = 20). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). Schematics of the NEAT1_2 configuration in the WT and Δ5′ mutant. Source data are available online for this figure. Source Data for Figure 2 [embj2020107270-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Characterization of NEAT1 mutants with deletions in the NEAT1_2 5′ terminal regions Quantitation of the relative expression levels of NEAT1_1 and NEAT1_2 by RT–qPCR in HAP1 WT, Δ5′, and Δ5′/ΔPAS cells treated with MG132 (5 μM for 6 h). Data are represented as mean ± SD (n = 3). (upper) Detection of NEAT1_2 by smFISH (magenta) in WT, Δ0–0.8 kb, Δ5′, and Δ5′/ΔPAS kb cells treated with MG132 (5 μM for 6 h). Nuclei were stained with DAPI. Scale bar, 10 nm. (lower) Quantitation of area and sum intensity per paraspeckle in each cell line (WT: n = 839, Δ0–0.8 kb: n = 845, Δ5′: n = 535, and Δ5′/ΔPAS: n = 652). (*P = 0.0420, ****P < 0.0001, compared with WT: Kruskal–Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the shell in WT, Δ0–0.8 kb, and Δ5′ cells treated with MG132 (5 μM for 6 h) by SRM analyses (WT: n = 167, Δ0–0.8 kb: n = 77, Δ5′: n = 48). (upper panels) EM observations of the paraspeckles in MG132-treated (5 μM for 17 h) WT and Δ5′ cells using the NEAT1_D2 probe. Scale bar, 100 nm. (bottom, left) Graph showing the proportion of localization (three layers: outer, middle, and inner layers [black circles indicate the boundaries]) of NEAT1_D2 probes (WT: 307 gold particles, Δ5′: 167 gold particles) within the paraspeckles in HAP1 Δ5′ cells. (bottom, right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in WT (n = 22) and Δ5′ cells (n = 15). (***P = 0.0001, ****P < 0.0001, compared with WT: Mann–Whitney test (two-tailed)). Each box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top). Quantitation of the relative expression levels of NEAT1_1 and NEAT1_2 by RT–qPCR in HAP1 WT and Δ0–2.8 kb cells. Data are represented as mean ± SD (n = 3). Detection of the paraspeckles by smFISH in WT and Δ0–2.8 kb cells. Scale bar, 10 nm. (left) The paraspeckles in Δ5′/ΔPAS cells treated with MG132 treatment (5 μM for 6 h) detected with SRM by NEAT1_2k (green) and 3′ (magenta) FISH probes. Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in Δ5′/ΔPAS cells (n = 31). Source data are available online for this figure. Download figure Download PowerPoint As the truncated 3′ terminal region of NEAT1_2 was localized in the paraspeckle core in the Δ3′ mutant cells, we attempted to obtain the corresponding 5′ terminal deletion clone of NEAT1 with exclusive core localization of the 5′ end, by establishing the additional 5′ terminal deletion cell line, NEAT1 Δ0–2.8 kb. However, in this cell line, NEAT1_2 was not expressed at all; hence, paraspeckles were not formed (Fig EV2E and F), precluding further analysis. NEAT1_1 overlaps with the 5′ terminal region of NEAT1_2, and it is impossible to distinguish these two transcripts by fluorescence in situ hybridization (FISH). Thus, to validate the configuration of the 5′ end of NEAT1_2 in the Δ5′ mutant cells, we established the Δ5′/ΔPAS cell line, which lacks a polyadenylation signal (PAS) for NEAT1_1 production. In this cell line, NEAT1_1 expression was reduced (Fig EV2A). The localization of the 5′ region of NEAT1_2 was still random within the paraspeckles, as observed in the Δ5′ mutant cells (Fig EV2G), suggesting that NEAT1_1 did not affect the arrangement of NEAT1_2 observed above. Thus, 5′ terminal deletion triggers the internalization of the 5′ truncated end of NEAT1_2. Simultaneous deletion of the 5′ and 3′ terminal domains of NEAT1_2 causes random distribution of NEAT1_2 within the paraspeckles As we had identified the NEAT1_2 RNA domains required for the shell localization of the 5′ and 3′ regions of NEAT1_2, we next investigated how the deletion of both the domains, 0–1.9 kb and 16.6–22.6 kb, influenced the NEAT1_2 spatial organization. We established a Δ5′/Δ3′ (Δ0–1.9 kb/Δ16.6–22.6 kb) mutant cell line, in which NEAT1_2 was expressed comparable to that in WT cells and no paraspeckle assembly defects were observed (Figs 3A and EV3A and B). In contrast to the highly ordered core-shell NEAT1_2 organization of the paraspeckles in WT cells, SRM observations clearly showed that the core-shell organization of the NEAT1_2 was totally lost in the Δ5′/Δ3′ mutant cells (Fig 3B and C). In addition, the 5′ and 3′ terminal regions of NEAT1_2 occupied different spaces within the paraspeckle, which might reflect the bundles of NEAT1_2 RNPs (West et al, 2016) and/or the hydrophilic nature of the NEAT1_2 5′ and 3′ regions to gather within the hydrophobic core. Additionally, EM analyses revealed that the 5′ terminal region was detected equally in both the shell and core of the paraspeckles, while the 3′ terminal region was almost randomly distributed with a slight tendency toward core localization (Fig 3D and E). These data were confirmed by SRM of the Δ5′/Δ3′/ΔPAS mutant cell line, which has reduced NEAT1_1 expression compared with the WT (Fig EV3C and D). Together with the data shown in Figs 1 and 2, these data suggested that both the 5′ and 3′ domains were essential for localizing both the ends of NEAT1_2 in the shell of paraspeckles. Figure 3. Deletion of both the 5′ and 3′ terminal domains of NEAT1_2 causes random distribution of both the 5′ and 3′ ends of NEAT1_2 within the paraspeckle The schematics of the WT and the mutant (Δ5′/Δ3′) with deletion of the 5′ and 3′ terminal regions are shown as Fig 1A. The positions of the NEAT1 probes used in SRM (blue) and EM (orange) are shown. SRM images of the paraspeckles in MG132-treated (5 μM for 6 h) HAP1 NEAT1 Δ5′/Δ3′ cells detected by NEAT1_2k (green) and NEAT1_15k FISH probes (left, magenta) or NONO immunofluorescence (IF; right, magenta). Scale bar, 500 nm. Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ (upper; WT: n = 41, Δ5′/Δ3′ kb: n = 44) and 3′
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