Fig 2: Formation of CoAZ foci depends on paraspeckles but not RNA. (A) HeLa cells were transiently transfected with the RFP-CoAZ and GFP-PSF expression vectors. Images were recorded at the indicated times after medium change (6 h post-transfection) using time-lapse fluorescence microscopy. Merged images are shown in the bottom row. (B) HeLa cells were transiently cotransfected with siRNA targeting PSF (siPSF), hnRNP K (sihnRNP K), NEAT1 (siNEAT1) or luciferase (siLuc, control) and the FLAG-CoAZ expression vector. Immunoblotting analysis was performed using antibodies against PSF, hnRNP K, FLAG, a-Tubulin and GAPDH. RT-PCR was performed to detect NEAT1, NEAT1_2 and GAPDH. Indirect immunofluorescence was performed using antibodies against FLAG, PSF and hnRNP K. FISH was performed with the NEAT1 probe. Merged images including Hoechst staining are shown at right. (C) HeLa cells were transfected with the FLAG-CoAZ expression vector for 24 h and then mock treated or treated with RNase for 30 min before cell fixation. Indirect immunofluorescence using anti-FLAG and anti-PSF was performed as in panel (B). Merged images including Hoechst (blue) staining are shown. (D) FRAP was performed in HeLa cells transiently expressing GFP-CoAZ at 25°C. Images show a representative GFP-CoAZ focus (circled in red) before bleaching and at the indicated times post-bleaching. The graph shows the fluorescence recovery of GFP-CoAZ as in Figure 3A. Scale bar represents 10 µm.

Fig 3: CoAZ is localized in close proximity to paraspeckles. (A) HeLa cells were transiently transfected with the vector expressing FLAG-tagged CoAZ, CoAZ-A0 or CoAZ-A25. Immunofluorescence was performed using anti-FLAG (green) and anti-PSF, anti-SC35 or anti-CoAA (red). Fluorescence in situ hybridization (FISH) was performed with the NEAT1 probe (red). Insets show higher magnification (3×) of selected FLAG-CoAZ foci. Merged images are shown in the right-most column. (B) FLAG-CoAZ (red) and GFP-PSF (green) were transiently expressed in HeLa cells. FLAG-CoAZ was detected by indirect immunofluorescence using anti-FLAG at low and high laser power. The merge of the high-power FLAG-CoAZ (red) and GFP-PSF (green) is shown in the lower-left panel, and a further merge with the Hoechst (blue) image is shown in the lower-middle panel. In merge, the line (with an arrowhead) indicates the fluorescence intensity scanning direction. The inset shows a higher magnification (3×) of the selected CoAZ/PSF foci.

Fig 4: CoAZ acts as a splicing regulator with activity distinct from that of RBM4. (A) Schematic diagram of the exon composition of RBM4a, CoAA and CoAZ. (B) The mock, FLAG-tagged RBM4 or CoAZ expression vectors were cotransfected with the E1a splicing reporter into HeLa cells or with the Kif1b into HEK293 cells. E1a splicing products were amplified by RT-PCR, fractionated on polyacrylamide gels and detected with 32P-labeled-specific probes in immunoblots (2). Kif1b splicing was analyzed as in Figure 3B. The relative efficiencies of distal 5' splice site utilization (9S/total) in E1a and exon inclusion of Kif1b are the averages (Avg) and SD of three independent experiments.