LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation (2019)
Figure 6

Figure 6. The HSATIII lncRNA accelerates CLK1-dependent re-phosphorylation of SRSF9

A Localization of SRSF9 with HSATIII during thermal stress and recovery. Scale bar: 10 μm.

B Recovery phase-specific colocalization of CLK1 and SRSF9. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and exposed to thermal stress (42°C for 2 h) followed by recovery (37°C for 1 h). FLAG-CLK1 and SRSF9 were co-stained using anti-FLAG and anti-SRSF9 antibodies, respectively, and the nuclei were stained with DAPI. Scale bar: 10 μm.

C Box plot of colocalization correlation coefficients between SRSF9 and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum are the ends of the whiskers.

(n=20 nuclei for each condition). *p<0.05 (Mann-Whitney U-test).

D Scheme of sampling references for analysis of the phosphorylation states of SRSF9 in (F). The CLK1 inhibitor KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The numbers correspond to the lane numbers in (F).

E Conditions tested in the time course analysis of the phosphorylation states of SR proteins. HeLa cells were transfected with a HSATIII ASO or control SO, cultured for 16 h, exposed to thermal stress (42°C for 2 h), and cultured at 37°C for the indicated period. F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively.

G Quantification of the data shown in (F). Graphs show the time-course changes in four representative SRSF9 phosphorylated forms in (F). Relative levels of band intensity were calculated as the ratio of each band to α-tubulin and were normalized to those of control at 37°C (bands (a) and (b)) and 42°C (bands (c) and (d)). Since band (d) partially overlapped with the other band (asterisk in (F)) at 37°C and recovery 4h, band (d) at those time points was not measured. Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).

H The re-phosphorylation status of SRSF9 within nSBs. Also, see Figure EV4G. Phosphorylated (arrows) and hypo- and de-phosphorylated (arrowheads) SRSF9 in the nSB fractions (lanes 4, 6, and 8) were analyzed by western blotting on Phos-tag gels. KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The lower panel shows a shorter exposure image of the phosphorylated forms. The asterisks indicate bands different from band (a) or (d). Input: 5%.

I Quantification of the data shown in (H). Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS-PAGE, and were normalized to those at 42°C. Data are shown as the mean±SD (n=4) (Dunnett's multiple comparison test) .