Extended Data Fig. 4: Effects of SFPQ deletion or mutations on interactions between SFPQ and the other DBHS family proteins and PS assembly.
a, Graph predicting PLD and IDR of SFPQ using PLAAC (upper) and PONDR (lower), respectively. b, Amino acid sequence of SFPQ. PLD is highlighted in red. c,d, Enrichment or depletion patterns of individual amino acids in SFPQ PLD detected by COMPOSITION PROFILER (refer to http://www.cprofiler.org). n = 100,000 bootstrap iterations. Data were compared using two-sample t-test (two-sided). e, Immunoblotting of Flag-tagged MCP-SFPQ WT and mutants in the mini-NEAT1 and mini-NEAT1/6Ă—MS2@8.2 kb cells (Fig. 4b, c). GAPDH is a control. MW are shown on the right. f, PSs (NEAT1_5′), the NSs (SON), and SFPQ in mini-NEAT1/6Ă—MS2@8.2 kb cells expressing MCP-SFPQ WT or ∆RRM2/NOPS and in HAP1 WT and mini-NEAT1 cells treated with MG132 (5 μM for 6 h). g, Quantification of fluorescence intensity ratio (SFPQ/NEAT1) as observed in f (n = 30). h, Proportion of the cells with PSs segregated from NSs in f. mini-NEAT1/6Ă—MS2@8.2 kb: n = 58 (MCP-SFPQ WT), 53 (∆RRM2/NOPS). i, Co-IP of MCP-SFPQ WT and mutant proteins with HA-SFPQ. Immunoblotting was performed to detect HA-tagged SFPQ proteins in the co-IP samples. GAPDH is a negative control. j, Quantification of the data shown in i. Values (HA/FLAG) are mean ± SD of three independent experiments. Data were compared using Kruskal-Wallis test (P = 0.03). k, PS formation with transfection of MCP-SFPQ WT and mutant proteins into m13–16.6 kb/6 Ă— MS2BS or m13–16.6 kb cells with MG132 treatment (5 μM for 6 h). The rescued PSs were visualized by NEAT1 RNA FISH (green) and IF of Flag-tagged MCP-SFPQ WT and mutants (magenta). l, PS sizes observed in k. m13-16.6 kb/6Ă—MS2BS: n = 78 (WT), 54 (ΔRRM2/NOPS), 65 (ΔCC), 62 (ΔPLD), 78 (Q to G), 66 (P to A partial); m13-16.6k: n = 58 (WT), 64 (ΔRRM2/NOPS), 53 (ΔCC), 62 (ΔPLD), 60 (Q to G), 56 (P to A partial). g,l, Each box plot shows the median (inside line), 25th-75th percentiles (box bottom to top), and minimum-maximum values (whisker bottom to top). Data were compared using Kruskal-Wallis ANOVA and post hoc Dunn’s multiple comparison test. Scale bar, 10 μm (f,k). Source numerical data and unprocessed blots are available in source data.
