Fig 2: Co-expression of RBM5 and InlP stimulates the redistribution of SC35-speckles at large deformed nuclear condensates. (A) Left, representative phase contrast and immunofluorescence confocal microscopy images of HeLa cells co-transfected with RBM5-Myc and InlP-HA (three examples are shown, with RBM5 condensates increasing in size); right, localization profile of RBM5 and InlP assessed with a line scan (white lines in the merged images) whose fluorescence intensity is plotted in red for RBM5, in green for InlP and in black for RBM5 granules; co-localization between RBM5 and SC35 is observed in I II, and III. (B) Nuclei with RBM5 speckles or RBM5 granules were scored in RBM5-expressing HeLa cells, in absence (black bars) or presence (grey bars) of InlP. Histograms represent percentage of each category as mean ± SD of 3 independent experiments. Statistical significance determined by ANOVA. (C,D) Representative phase contrast and immunofluorescence confocal microscopy images of HeLa cells co-expressing RBM5-Myc and GFP-InlP, immunolabeled with SC35 and RBM5 antibodies (A) or SC35 antibody (B). A nucleus with RBM5-condensates formed in presence of GFP-InlP is shown at higher magnification in (D). Arrows point to sites that show potential events of coalescence or fission. Scale bars: 10 µm.

Fig 3: InlP stimulates formation of RBM5-associated nuclear granules. (A) Representative micrographs of the three subtypes of RBM5-Myc-expressing cells, with the RBM5-Myc immunostaining on top and the phase-contrast image below (bars: 10 µm). A higher magnification of each squared region is shown in the upper left corner (bars: 2 µM). The percentage of each cell subtype is indicated as a mean ± SD of 3 independent experiments, with n = 30 cells per replicate. (B) Left, representative phase contrast and immunofluorescence confocal microscopy images of transfected RBM5-Myc and SC35, in presence or absence of InlP-HA. Three subtypes of RBM5-expressing HeLa cells are shown: a cell with a nucleus containing RBM5-speckles (first row); a cell with a nucleus containing RBM5 granules (second row); a cell with a nucleus containing RBM5 granules in presence of InlP (third row). Scale bars: 10 µm. A higher magnification of each boxed region in the Merge/Phase image is shown in the upper left corner (Scale bars: 2 µm); Right, localization profile of RBM5 and SC35 assessed with a line scan (white lines in the merged images) whose fluorescence intensity is plotted in red for RBM5, in green for SC35 and in black for RBM5 granules; faint co-localization between RBM5 and SC35 is observed in I and II, while a more extensive colocalization is measured in III and IV. (C) Nuclei with RBM5 granules were scored in HeLa cells transfected RBM5-Myc or co-transfected with RBM5-Myc and InlP-HA or Lmo2027-HA. Histograms represent percentage of each category as mean ± SD of 3 independent experiments. Statistical significance determined by ANOVA.

Fig 4: The OCRE domain of RBM5 is required for the functional interaction with InlP. (A) Top, schematic representation of RMB5 full length and mutants used in immunoprecipitation experiments. Bottom, Myc immunoprecipitation (IP) of RBM5-Myc-FLAG or RBM5 mutants (RBM5∆OCREMyc-FLAG; RBM5∆RRMMyc-FLAG) in HEK293-FT cells co-expressing or not InlP-HA-FLAG. The FLAG antibody was used to detect RBM5 and InlP on the same immunoblot for comparative purposes. The arrow points to the impaired interaction between RBM5∆OCRE and InlP. (B) Percentage of cells showing aberrant nuclei after transfection of RBM5, RBM5∆RRM, RBM5∆OCRE, in presence or absence of InlP. Data are mean ± SD from at least three independent biological replicates. Statistical significance determined by ANOVA.