Fig. 5: Accumulation of active spliceosomes and poly(A)+ RNA in unique splicing clusters of human RP13 photoreceptors.

A Western blotting of the whole extracts from the KiOs, RPE and ROs tissues for the indicated splicing proteins. The beta-actin was used as a loading control for quantification; (B) Graphs showing quantification of p-SF3B1 (245.9 ± 4.4) and p-PRPF31 (145.4 ± 22.9) Western blot bands in RP13 RPE relative to the Cas9 controls (100.0 ± 0.0). The differences between RP13 and controls (two samples in each group) were only significant for p-SF3B1 (P-value = 0.0009), (***P-value < 0.001, two-tailed t test); (C) In photoreceptor cells active spliceosomes marked with p-SF3B1 (red) are localised in SC35 (green) clusters adjacent to DAPI-stained DNA foci (blue) at the periphery of the nucleus. The insets show the magnification of the selected regions. The experiment was repeated three times, independently. Scale bar 10 µm; (D) Accumulation of active spliceosomes (p-SF3B1, red) in splicing clusters (SC35, green) of RP13 photoreceptors. The insets show the magnification of the selected regions. The experiment was repeated three times, independently. Scale bar 10 µm; (E) Quantification of the intensities of p-SF3B1 clusters in RP13 versus Cas9 control photoreceptors (n ≥ 120 cells analysed) in (D); (F) Increased levels of poly(A)+ RNA (red) within the splicing clusters (SC35, green) in RP13-photoreceptors, monitored by RNA FISH followed by immunostaining for SC35. The insets show the zoom of the selected regions. The arrow indicates RNAs localised to the nuclear periphery in the control. The experiment was repeated two times, independently. Scale bar 10 µm; (G) Quantification of the co-localisation of poly(A)+ RNA with SC35-positive clusters in RP13 versus RP13-Cas9-photoreceptors (n ≥ 150 cells analysed) in (F); (H) Immunofluorescence imaging of photoreceptors labelled with RNAPII (green), p-SF3B1 (red) and counterstained with DAPI (blue) showing concentration of RNAPII in the splicing clusters in photoreceptors. The inset shows the magnification of the selected region. The arrow indicates RNAPII localised to the nuclear periphery. The experiment was repeated three times, independently. Scale bar 10 µm; (I) Schematic representation of the nuclear architecture in human photoreceptors showing their unique chromatin organisation and splicing clusters. The schematic was created with BioRender.com.