Fig 1: Generation and characterization of a stable HeLaGFP-NONOcell line.A, schematic representation of a PTRE3G–tagGFP–NONO plasmid that was transfected in HeLa cells constitutively expressing the Tet-On 3G transactivator to generate a stable HeLaGFP-NONO cell line. Expression of GFP-NONO is induced only in the presence of Dox. B, Western blot of protein lysates extracted from HeLaGFP-NONO cells that have been induced with a range of concentrations of Dox for 24 h, showing that different concentrations of Dox gave different levels of expression of GFP-NONO but endogenous NONO remained constant. Molecular weight markers (kDa) are at left. Anti-NONO monoclonal antibody was used to probe for GFP-NONO (82 kDa) and endogenous NONO (55 kDa). Beta Actin (45 kDa) was used as the loading control. C, Western blot of protein lysates extracted from HeLaGFP-NONO cells that have been induced with 50 ng/ml of Dox over different times. Induction with Dox for both 24 h and 48 h gave similar expression level of GFP-NONO. Proteins were detected as in (B). ∗denotes some sporadic cross-reactivity of the NONO antibody with endogenous PSPC1. D, Western blot of protein lysates extracted from HeLaGFP-NONO cells that have been induced with 50 ng/ml of Dox for 24 h, followed by removal of Dox for different times ranging from 0 to 48 h. Upon withdrawal of Dox, detectable amount of GFP-NONO gradually decreased over time. Proteins were detected as in (B). Sample not induced with Dox serves as negative control in lane 1. ∗denotes some sporadic cross-reactivity of the NONO antibody with endogenous PSPC1. Panels (B–D) are single representative blots from multiple repeated experiments (minimum n = 3). E, widefield fluorescent micrograph of several fixed HeLaGFP-NONO cells showing bright and distinct paraspeckles, arrows, with nucleoplasmic background signal. F, representative fluorescence micrographs of HeLaGFP-NONO cells either without Dox induction (left) or following Dox induction (right) show that GFP-NONO localizes with NEAT1 in paraspeckles in an identical manner to endogenous NONO. Top to bottom: DAPI; NEAT1 FISH to detect paraspeckles; anti-NONO immunofluorescence to detect endogenous NONO (left) and a mixture of endogenous NONO and Dox-induced GFP-NONO (right); and GFP-NONO fluorescence. Arrows indicate colocalization in paraspeckles. The scale bar represents 10 μm. G, merged fluorescence images of selected channels from (F), showing at left that in the absence of Dox, endogenous NONO (green) colocalizes with NEAT1 (red) in paraspeckles (yellow). Right panel shows in the presence of Dox, GFP-NONO (green) colocalizes with NEAT1(red) in paraspeckles (yellow). Top panels show overview, middle panel shows zoomed in paraspeckle clusters corresponding to white boxes in top panel. Bottom panel shows line-scan profile of the red and green fluorescence signals across the line as displayed in the middle panel. Western blots and micrographs are representative examples of >3 biological replicates. DAPI, 4′,6-diamidino-2-phenylindole; Dox, Doxycycline.
Fig 2: Assessing DBHS protein partner swapping by Ni-affinity pull downs, analytical size-exclusion chromatography, and structural comparisons.A, polyacrylamide gel of H6SFPQ and NONO, showing pull down of NONO. 1: H6SFPQ, 2: NONO, 3: H6SFPQ Ni-affinity flow through, 4: H6SFPQ Ni-affinity elution, 5: NONO Ni-affinity flow-through, 6: NONO Ni affinity elution, 7: H6SFPQ and NONO incubated 1:1 at ambient temperature flow-through, 8: H6SFPQ and NONO incubated 1:1 at ambient temperature elution, 9: H6SFPQ and NONO incubated 1:1 at 37 °C flow through, 10: H6SFPQ and NONO incubated 1:1 at 37 °C elution. B, analytical size-exclusion profile of MBP-NONO (dashed blue), SFPQ (dashed red), and MBP-NONO/SFPQ heterodimer (solid black). C, cartoon representation of SFPQ homodimer (4wii, cyan) mixed with structured NONO homodimer (5ifm, green) resulting in NONO-SFPQ heterodimer (7lrq). D, view down the longitudinal axis of the coiled-coil reveals structural differences in the NOPS domain (a5 helix) and distal coiled-coil (a6 helix) on both sides of the dimer. Top: NONO-SFPQ heterodimer superposed with NONO homodimer; bottom: NONO-SFPQ superposed with NONO-PSPC1 heterodimer. E, view from below (down the 2-fold axis of the dimer) shows that, relative to NONO-SFPQ, the RRM1 domains of both the other NONO-containing dimers are rotated ~10° to 20° clockwise. Proteins are shown in cartoon representation, with NONO colored green, SFPQ cyan, and PSPC1 orange. All six dimers of the NONO homodimer are superposed in both orientations in (D) and (E). Helices are shown as cylinders in (E).
Fig 3: 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
Fig 4: DBHS protein family members form homodimers and heterodimers dimers. Each member contains a core conserved region flanked by variable regions of low sequence complexity. A, schematic alignment of the three human DBHS proteins, SFPQ, NONO, and PSPC1, with relevant domain detail highlighted. B, X-ray crystal structure showing the dimer formed by the conserved DBHS region (SFPQ homodimer; PDB 4wij; residues 276–598). The domains of one dimer partner (black outline) are labeled. PDB, Protein Data Bank.
Fig 5: 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
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