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Shell protein composition specified by the lncRNA NEAT1 domains dictates the formation of paraspeckles as distinct membraneless organelles

Nature Cell Biology volume 25, pages 1664–1675 (2023)Cite this article

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Abstract

Many membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Although these MLOs co-exist in cells, how they maintain their independence without coalescence or engulfment remains largely unknown. Here, we investigated the molecular mechanism by which paraspeckles with core–shell architecture scaffolded by NEAT1_2 long noncoding RNAs exist as distinct MLOs. We identified NEAT1 deletion mutants that assemble paraspeckles that are incorporated into nuclear speckles. Several paraspeckle proteins, including SFPQ, HNRNPF and BRG1, prevent this incorporation and thus contribute to the segregation of paraspeckles from nuclear speckles. Shell localization of these proteins in the paraspeckles, which is determined by NEAT1_2 long noncoding RNA domains, is required for this segregation process. Conversely, U2-related spliceosomal proteins are involved in internalizing the paraspeckles into nuclear speckles. This study shows that the paraspeckle shell composition dictates the independence of MLOs in the nucleus, providing insights into the importance of the shell in defining features and functions of MLOs.

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Fig. 1: Mini-PSs are incorporated into NSs.
Fig. 2: A subset of PSPs are mislocalized in mini-PSs.
Fig. 3: Artificial tethering of SFPQ, HNRNPF and BRG1 to the shells induces segregation of mini-PS from NSs.
Fig. 4: The dimerization, oligomerization and prion-like domains of SFPQ are required for the mini-PS segregation from NSs.
Fig. 5: U2 snRNP-related proteins are major interacting proteins of the mini-NEAT1 RNA domain responsible for incorporation into NSs.
Fig. 6: Artificial shell tethering of U2 snRNP components induces the internalization of PSs into NSs.

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Data availability

The MS data were deposited in the Japan Proteome Standard repository (jPOST) under the ID JPST002268. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank M. Okamura, C. Fujikawa and A. Kubota for technical support and A. Marshall, A. H. Fox, C. S. Bond, B. Dyakov, A. C. Gingras, T. Yoda, M. Murakami and members of the Hirose laboratory for valuable discussions. This research was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (to T. Yamazaki (19K06479, 19H5250, 21H00253, 22H02545), to S.A. (21H05736, 22H02225), to T. Yamamoto (20H05934, 21H00241, 21K03479), to T.H. (20H00448, 20H05377, 21H05276, 22K19293)), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (T. Yamazaki), the Naito Foundation (T. Yamazaki), the Takeda Science Foundation (T. Yamazaki), JST CREST (JPMJCR20E6) to T.H. and an AMED grant (JP21ae0121049) to T.H. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Hiro Takakuwa, Tomohiro Yamazaki.

Authors and Affiliations

  1. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan

    Hiro Takakuwa, Tomohiro Yamazaki, Naoko Fujiwara & Tetsuro Hirose

  2. Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

    Hiro Takakuwa & Hyura Yoshino

  3. UMS 3655, AMMICA, Gustave Roussy, Villejuif, France

    Sylvie Souquere

  4. Department of Proteomics, National Cancer Center Research Institute, Tokyo, Japan

    Shungo Adachi

  5. Institute for Chemical Reaction Design and Discovery, Hokkaido University, Sapporo, Japan

    Tetsuya Yamamoto

  6. Cellular and Molecular Biotechnology Research Institute, National Institute for Advanced Industrial Science and Technology, Tokyo, Japan

    Tohru Natsume

  7. Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

    Shinichi Nakagawa

  8. Centre National de la Recherche Scientifique, UMR-9196, Gustave Roussy, Villejuif, France

    Gerard Pierron

  9. Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Japan

    Tetsuro Hirose

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Contributions

H.T., T. Yamazaki and T.H. conceived and designed this study. H.T. and T. Yamazaki conducted most of the experiments. S.S. and G.P. performed EM analyses. S.A. and T.N. performed MS analyses. N.F. contributed to RNA-seq data analysis and manuscript editing. T. Yamamoto contributed to theoretical interpretations of the experimental data. H.Y. and S.N. contributed to the SRM analyses. H.T., T. Yamazaki and T.H. wrote the manuscript.

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Correspondence to Tomohiro Yamazaki or Tetsuro Hirose.

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Extended data

Extended Data Fig. 1 Mini-PSs are present within NSs, although NEAT1 transcription occurs outside of NSs.

a, Schematics of the NEAT1_2 configuration in the WT-PS and mini-PS. b, SRM images of PSs (RNA-FISH with NEAT1_5′ probe) and NSs (SRRM2 IF) in WT and mini-NEAT1 mutant cells in the absence of MG132. Scale bar, 500 nm. c, Graph showing the proportion of segregated or incorporated PSs in b. Data were collected from two independent experiments. WT: n = 167, mini-NEAT1: n = 210. d, SRM images of the PSs (RNA-FISH with NEAT1_5′ probe) and NSs (SRRM2 IF) in WT and another mini-NEAT1 mutant clone treated with MG132 (5 μM for 6 h). Scale bar, 10 μm. e, PSs and NSs were detected by smFISH with NEAT1_5′ probes (magenta) and SON IF (green) in the presence of MG132 (5 μM for 6 h). Nuclei were stained with DAPI. Line profiles of the PSs and NSs are shown (right). f, SRM images of PSs (smFISH with NEAT1_5′ probe) and NSs (SON IF) in WT and mini-NEAT1 cells treated with MG132 (5 μM for 6 h). Scale bar, 500 nm. g, PSs and NSs in WT and mini-NEAT1 cells were detected by RNA-FISH with NEAT1_5′ probe and MALAT1 probe (green and magenta, respectively) in the presence of MG132 (5 μM for 6 h). Nuclei were stained with DAPI. Line profiles of the PSs and NSs are shown (right). Scale bar, 10 μm. h, SRM images of PSs (RNA-FISH with NEAT1_5′ probe) and NSs (RNA FISH with MALAT1 probe) in WT and mini-NEAT1 cells treated with MG132 (5 μM for 6 h). Scale bar, 500 nm. i, EM observation of multiple, but unconnected, mini-PSs within an NS. NONO localization was detected as gold particles. Dotted magenta circles indicate the mini-PS position. Dotted black circles indicate the NS position. Scale bar, 500 nm. j, PSs and other nuclear bodies in WT and mini-NEAT1 cells treated with MG132 (5 μM for 6 h). PSs were visualized by NEAT1 RNA-FISH with NEAT1_5′ probe (green). Nuclear bodies (parentheses) were visualized by IF of marker proteins (magenta). Nuclei were stained with DAPI. Scale bar, 10 μm. Source numerical data are available in source data.

Source data

Extended Data Fig. 2 A subset of PSPs are similarly localized in WT-PSs and mini-PSs.

a, Immunoblotting to examine the expression levels of various PSPs (BRG1, SFPQ, PSPC1, NONO, HNRNPH1, HNRNPF, and TDP-43) in HAP1 WT and the NEAT1 deletion mutant cells in Fig. 2a. Cells were treated with MG132 (5 μM for 6 h). GAPDH was used as a control. b, Confocal observation of the PSs and various PSPs in HAP1 WT and mini-NEAT1 mutant cells treated with MG132 (5 μM for 6 h). PSs and PSPs were visualized by RNA-FISH using NEAT1_5′ probes (green) and IF (magenta). Nuclei were stained with DAPI. Scale bar, 10 μm. c, Quantification of fluorescence intensity ratio (PSPs/NEAT1) in the HAP1 WT (blue) and mini-NEAT1 (pink) cells as observed in b (all samples: n = 30). Each box plot shows the median (inside line), 25th-75th percentiles (box bottom to top), and minimum-maximum values (whisker bottom to top). Data were compared using Mann-Whitney U-test (two-sided). If the statistical test showed no significant difference (P > 0.05), it is not specified in the figure. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Reciprocal recruitment of SFPQ, HNRNPF, and BRG1 by their tethering.

a, Genotyping PCR at the mini-NEAT1 locus. Electrophoresis of the PCR products confirmed the 6×MS2 knock-in. The primers used are listed in Supplementary Table 2. b, Immunoblotting of Flag-tagged MCP-PSPs in the mini-NEAT1 and mini-NEAT1/6×MS2@8.2 kb cells (Fig. 3b and 3c). GAPDH is a control. Molecular weight markers (MW) are shown on the right. c, Immunoblotting of Flag-tagged MCP-PSPs in the mini-NEAT1, mini-NEAT1/6×MS2@8.2 kb, and mini-NEAT1/6×MS2@16.2 kb (Fig. 3b–d). GAPDH is a control. MW are shown on the right. d, Quantification of fluorescence intensity ratio (FLAG/NEAT1) in three mutant cells shown (n = 20). e–g, Quantification of fluorescence intensity ratio (PSPs/NEAT1) with transfection of MCP-PSPs into mini-NEAT1/6×MS2@8.2 kb cells. (e) SFPQ recruitment: n = 30 (MCP-GFPNLS), n = 30 (MCP-HNRNPF), n = 25 (MCP-BRG1), n = 30 (MCP-NONO), n = 30 (MCP-PSPC1). (f) HNRNPF recruitment: n = 30 (MCP-GFPNLS), n = 30 (MCP-SFPQ), n = 30 (MCP-BRG1). (g) BRG1 recruitment: n = 30 (MCP-GFPNLS), n = 30 (MCP-SFPQ), n = 30 (MCP-HNRNPF). h, PSs and NSs with transfection of MCP-NONO or PSPC1 into mini-NEAT1/6×MS2@8.2 kb (upper) and mini-NEAT1 (lower) treated with MG132 (5 μM for 6 h). White boxes indicate the areas shown at a higher magnification. Scale bar, 10 μm. i, Recruitment of various PSPs to PSs in WT and mini-NEAT1, and ∆4–8 kb/16.6–22.6 kb, and ∆1–4 kb/16.6–22.6 kb mutants treated with MG132 (5 μM for 6 h). PSs and PSPs were visualized by RNA-FISH using NEAT1_5′ probes (green) and IF (magenta). Nuclei were stained with DAPI. Scale bar, 10 μm. j, Quantification of fluorescence intensity ratio (PSPs/NEAT1) in the WT and four mutants shown as observed in i (all samples: n = 30). For d–g,j, each box plot shows the median (inside line), 25th-75th percentiles (box bottom to top), and minimum-maximum values (whisker bottom to top). Data were compared using Kruskal-Wallis ANOVA and post hoc Dunn’s multiple comparison test. The statistical test showing no significant difference (P > 0.05) is not specified in the figure. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 Effects of SFPQ deletion or mutations on interactions between SFPQ and the other DBHS family proteins and PS assembly.

a, Graph predicting PLD and IDR of SFPQ using PLAAC (upper) and PONDR (lower), respectively. b, Amino acid sequence of SFPQ. PLD is highlighted in red. c,d, Enrichment or depletion patterns of individual amino acids in SFPQ PLD detected by COMPOSITION PROFILER (refer to http://www.cprofiler.org). n = 100,000 bootstrap iterations. Data were compared using two-sample t-test (two-sided). e, Immunoblotting of Flag-tagged MCP-SFPQ WT and mutants in the mini-NEAT1 and mini-NEAT1/6×MS2@8.2 kb cells (Fig. 4b, c). GAPDH is a control. MW are shown on the right. f, PSs (NEAT1_5′), the NSs (SON), and SFPQ in mini-NEAT1/6×MS2@8.2 kb cells expressing MCP-SFPQ WT or ∆RRM2/NOPS and in HAP1 WT and mini-NEAT1 cells treated with MG132 (5 μM for 6 h). g, Quantification of fluorescence intensity ratio (SFPQ/NEAT1) as observed in f (n = 30). h, Proportion of the cells with PSs segregated from NSs in f. mini-NEAT1/6×MS2@8.2 kb: n = 58 (MCP-SFPQ WT), 53 (∆RRM2/NOPS). i, Co-IP of MCP-SFPQ WT and mutant proteins with HA-SFPQ. Immunoblotting was performed to detect HA-tagged SFPQ proteins in the co-IP samples. GAPDH is a negative control. j, Quantification of the data shown in i. Values (HA/FLAG) are mean ± SD of three independent experiments. Data were compared using Kruskal-Wallis test (P = 0.03). k, PS formation with transfection of MCP-SFPQ WT and mutant proteins into m13–16.6 kb/6 × MS2BS or m13–16.6 kb cells with MG132 treatment (5 μM for 6 h). The rescued PSs were visualized by NEAT1 RNA FISH (green) and IF of Flag-tagged MCP-SFPQ WT and mutants (magenta). l, PS sizes observed in k. m13-16.6 kb/6×MS2BS: n = 78 (WT), 54 (ΔRRM2/NOPS), 65 (ΔCC), 62 (ΔPLD), 78 (Q to G), 66 (P to A partial); m13-16.6k: n = 58 (WT), 64 (ΔRRM2/NOPS), 53 (ΔCC), 62 (ΔPLD), 60 (Q to G), 56 (P to A partial). g,l, Each box plot shows the median (inside line), 25th-75th percentiles (box bottom to top), and minimum-maximum values (whisker bottom to top). Data were compared using Kruskal-Wallis ANOVA and post hoc Dunn’s multiple comparison test. Scale bar, 10 μm (f,k). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 5 U2 snRNP components are included in both NS and PS proteomes.

a, Confocal observation of PSs, BRG1, and SFPQ in HAP1 WT, mini-NEAT, and m9.8–16.6 kb mutant cells treated with MG132 (5 μM for 6 h). PSs were visualized by NEAT1 RNA-FISH with NEAT1_5′ probe (white). BRG1 (green) and SFPQ (magenta) were visualized by IF. Nuclei were stained with DAPI. Scale bar, 10 μm. b, Quantification of fluorescence intensity ratio (PSPs/NEAT1) observed in a (all samples: n = 30). Each box plot shows the median (inside line), 25th-75th percentiles (box bottom to top), and minimum-maximum values (whisker bottom to top). Data were compared using Kruskal-Wallis ANOVA and post hoc Dunn’s multiple comparison test. If the statistical test showed no significant difference (P > 0.05), it is not specified in the figure. c,d, Venn diagram showing the overlap between 8–9.2 kb and 9.2–9.8 kb of RNA pulldown (c), or between NEAT1 HyPro-MS39 and SC35 TSA-MS38 (d). e, Top 10 GO:CC enrichment for proteins shared between NEAT1 HyPro-MS39 and SC35 TSA-MS38. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 SF3A and SF3B complex components bind to the 5′ terminal shell region of NEAT1, and the NEAT1_2 sequence contains multiple potential branch point sequences.

a, Genotyping PCR data are as in Extended data Fig. 3. b, SRM maximum projection images of PSs and NSs with transfection of MCP proteins into NEAT1/6×MS2@1.4 kb (left), NEAT1/6×MS2 @14 kb (middle), and WT (right) with MG132 treatment (5 μM for 6 h). White boxes indicate the areas shown at a higher magnification. Scale bar, 5 μm. c, Proportion of the cells with PSs segregated from NSs in b. NEAT1/6×MS2@1.4 kb @1.4 kb; n = 60 (SF3A3-MCP), 59 (MCP-PTBP1), NEAT1/6×MS2@1.4 kb @14 kb; n = 61 (SF3A3-MCP), 59 (MCP-PTBP1), WT; n = 62 (SF3A3-MCP), 64 (MCP-PTBP1). Data were compared using Fisher’s exact test (two-sided) and Bonferroni correction. d, Immunoblotting of Flag-tagged MCP proteins used in Fig. 6b, c and Extended Data Fig. 6b, c. GAPDH is a control. MW are shown on the right. e, SRM images of localization of SF3B1 within PSs in WT and mini-NEAT1 cells treated with MG132 (5 μM for 6 h) (upper). PSs were visualized by RNA FISH with NEAT1_5′ probe (green), and SF3B1 IF (magenta). Scale bar, 500 nm. Graph showing average fluorescence intensity of SF3B1 in the shell or core of PSs (lower) (n = 31). Each box plot shows the median (inside line), 25th-75th percentiles (box bottom to top), and minimum-maximum values (whisker bottom to top). Data were compared using Kruskal-Wallis ANOVA and post hoc Dunn’s multiple comparison test. f, U2 snRNA expression levels of the U2 ASO-transfected mini-NEAT1 mutant cells treated with MG132 (5 μM for 6 h) by RT-qPCR in Fig. 6f, g. Data are presented as mean ± SD (n = 3). Data were compared using one-way ANOVA and post hoc Tukey’s multiple comparison test. g, eCLIP data of SF3B4 (HepG2 and K562), SF3B1 (K562), SF3A3 (HepG2), U2AF2 (HepG2 and K562), SFPQ (HepG2), and NONO (K562) and HITS-CLIP data of HNRNPF (293 T)53 mapped on NEAT1_2. The number of SF1 consensus motifs (YNYURAY; Y = U or C, R = A or G, N = any nucleotide) present in the NEAT1_2 sequence is shown66. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 7 Shell protein composition, but not core protein composition, of the WT-PS or mini-PS dictates their localization to NS.

A summary of the shell or core tethering in the WT-PS (upper) and mini-PS (lower) is depicted.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Tables

Supplementary Tables 1–7.

Supplementary Video 1

3D visualization of PSs and NSs in HAP1 WT cells in the presence of MG132 (5 μM for 6 h), corresponding to Fig. 1e. PSs were visualized by NEAT1 RNA FISH with NEAT1_5′ probe (green), and NSs were visualized by SRRM2 IF (magenta).

Supplementary Video 2

3D visualization of PSs and NSs in mini-NEAT1 mutant cells in the presence of MG132 (5 μM for 6 h), corresponding to Fig. 1e. PSs were visualized by NEAT1 RNA FISH with NEAT1_5′ probe (green), and NSs were visualized by SRRM2 IF (magenta).

Source data

Figs. 1–6 and Extended Data Figs. 1–6

Statistical source data.

Figs. 2, 4 and Extended Data Figs. 2, 4, 6

Unprocessed western blots and/or gels.

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Takakuwa, H., Yamazaki, T., Souquere, S. et al. Shell protein composition specified by the lncRNA NEAT1 domains dictates the formation of paraspeckles as distinct membraneless organelles. Nat Cell Biol 25, 1664–1675 (2023). https://doi.org/10.1038/s41556-023-01254-1

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