Fig 1: Neat1 RNA in paraspeckle nuclear bodies: association with paraspeckle proteins and visualization by FISH.(A) Association of paraspeckle proteins with Neat1 RNA RNA Immuno-Precipitation (RIP) experiments (n=4 for each antibody) using antibodies directed against NONO, SFPQ, RBM14 or PSPC1 show the enrichment in Neat1 RNA obtained as compared to an irrelevant non-specific antibody (see Figure 2—source data 1 for primer sequences). **p<0.01 vs non-specific antibody. B. Visualization of Neat1 RNA by Fluorescence in situ Hybridization and confocal laser scanning microscope Left Panel: RNA-FISH shows the distribution of Neat1 RNA in a few distinct foci (arrows). The round aspect of the foci under confocal laser scanning microscope is shown in the insert in which assigned foci is enlarged. Scale bars: 1 µm Right Panel: The extent of the nucleus is shown with Hoechst staining. Foci containing Neat1 RNA localize within the nucleus sometimes in the close vicinity of nucleus boundaries. Scale bars: 5 µm. (C) Visualization of Neat1 RNA by Fluorescence in situ Hybridization and super resolution Left Panel: Conventional fluorescence microscopy of Neat1 RNA-FISH. The nucleus is outlined with hand-drawn dashed lines to indicate the nuclear periphery. Scale bars: 5 µm. Right Upper Panel: Enlargement of the foci assigned in left panel allows to show the poor resolution of paraspeckle under conventional fluorescence microscopy (in red). In white is the superimposed high-resolution image obtained after STORM analysis. Note that the size of paraspeckle after STORM analysis is strongly reduced. Scale bars: 0.5 µm. Right Bottom Panel: Enlargement of the STORM analysis shown in the upper panel with measurements of width (Wstorm=0.14 µm) and height (hstorm=0.17 µm). Note the elliptical shape of the foci analyzed. Scale bars: 0.1 µm.DOI: http://dx.doi.org/10.7554/eLife.14837.00410.7554/eLife.14837.005Figure 2—source data 1.Sequences of qPCR primers and oligonucleotides.DOI: http://dx.doi.org/10.7554/eLife.14837.005
Fig 2: Rhythmic expression and association of paraspeckle components.(A) Rhythmic expression of two paraspeckle proteins in pituitary GH4C1 cells The expression of PSPC1 and RBM14 is determined by Western Blot analysis over a 30 hr time period. Each data point (mean ± SD of three independent samples) represents the ratio of the depicted proteins to ATF2 and is expressed relative to the value obtained at ZT 30. Experimental values can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see also Figure 3—source data 1). (B) Rhythmic expression of the long noncoding Neat1 RNA. The expression of the lncRNA Neat1 is determined by RT-qPCR over a 40 hr time period. Primers used to allow the detection of both Neat1-1 and Neat1-2. Experimental values (n=4) expressed as a percent of the initial value obtained at ZT 0 can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see also Figure 3—source data 1). (C) Rhythmic association of paraspeckle proteins with Neat1 RNA RNA Immuno-Precipitation (RIP) experiments (n=4 for each antibody) are performed over a 30h time period. At each time point, the levels of Neat1 RNA determined after immuno-precipitation by the antibodies directed against NONO, SFPQ, RBM14 and PSPC1 were normalized relative to Neat1 RNA input levels and expressed as a percent of the value obtained at T0. Experimental values can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see also Figure 3—source data 2). (D–E) Rhythmic fluctuations of paraspeckle number Cells were arrested at four different times after the medium change and processed for FISH of Neat1 RNA. At each time point, 20 to 35 images from four wells of 100 000 cells obtained in two different experiments were acquired under a confocal microscope with a 40X objective. At each time point, the total number of paraspeckles per well and the mean number of paraspeckles per cell were calculated. **p<0.001 ****p<0.0001.DOI: http://dx.doi.org/10.7554/eLife.14837.00610.7554/eLife.14837.007Figure 3—source data 1.Cosinor analysis of the rhythmic expression pattern of paraspeckle components in GH4C1 cells.DOI: http://dx.doi.org/10.7554/eLife.14837.00710.7554/eLife.14837.008Figure 3—source data 2.Cosinor analysis of the rhythmic binding of the four paraspeckle-associated proteins on Neat1 RNA in GH4C1 cells.DOI: http://dx.doi.org/10.7554/eLife.14837.008
Fig 3: Influence of IRAlu elements inserted in 3'-UTR egfp mRNA.(A–C) Decrease in EGFP expression by insertion of IRAlus in the 3’ -UTR of egfp mRNA. IRAlus and Alu were PCR-amplified from the 3’-UTR of Nicn1 and then inserted separately into the 3’-UTR of egfp mRNA. GH4C1 cell lines, Alu-egfp and IRAlu-egfp cell lines were established by transfection of the indicated plasmids. (A) Representative example of fluorescence and corresponding bright field pictures taken 48 hr after platting of each cell line. Scale bars equal 50 µm. (B) Quantitative analysis of the percent of fluorescent cells in each cell line. Data are means ± SEM of 18 measures performed in 3 independent experiments. (C) Quantification of relative levels of eGFP investigated by western blotting with anti-GFP antibody in total protein extracts from the two cell lines. Tubulin was used as the loading control. Data are mean ± SEM of values obtained from three experiments in IRAlu-egfp cell line and are expressed as a percent of the corresponding value obtained in Alu-egfp cell line. (D) Nuclear and cytoplasmic egfp mRNA were quantified by qPCR in each cell line and normalized to the relative amount of gapdh mRNA (n=8 for each cell line). Ratio of nuclear versus cytoplasmic egfp mRNA levels are compared between IRAlu-egfp and Alu-egfp cell lines. (E) Enrichment in lncRNA Neat1 after RNA Immuno-Precipitation (RIP) with an antibody directed against PSPC1 relative to an irrelevant antibody (left panel) or after RNA pull-down with two different specific biotinylated oligonucleotides (S oligo 1 and S oligo 2) relative to a non-specific oligonucleotide (two right panels). The relative enrichment in lncRNA Neat1 obtained after either RIP (n=3 for each cell line) or RNA pull-down (n=6 for each cell line) is not statistically different in Alu-egfp versus IRAlu-egfp cell lines F. Enrichment in egfp mRNA after RNA Immuno-Precipitation (RIP) with an antibody directed against PSPC1 relative to an irrelevant antibody (left panel) or after RNA pull-down with two different specific biotinylated oligonucleotides (S oligo 1 and S oligo 2) relative to a non-specific oligonucleotide (two right panels). The relative enrichment in egfp mRNA obtained after either RIP (n=3 for each cell line) or RNA pull-down (n=6 for each cell line) is statistically higher in IRAlu-egfp versus Alu-egfp cell lines. *p<0.05 **p<0.01***p<0.001****p<0.0001.DOI: http://dx.doi.org/10.7554/eLife.14837.010
Fig 4: Localization by confocal microscopy of paraspeckle proteins in GH4C1 pituitary cells.Cells are grown on coverslips and labeled for immunofluorescence with antibody to SFPQ (green A, C), NONO (green B, D) PSPC1 (red A, B) or RBM14 (red C, D). SFPQ or NONO staining is merged with PSPC1 (A, B 3th column) or RBM14 (C, D 3th column). Nuclear staining by Hoechst for the same samples is added in 4th column. Arrows indicate punctate clusters in which two paraspeckle proteins overlap. Scale bars: 5 µm.DOI: http://dx.doi.org/10.7554/eLife.14837.003
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