Fig. 3: PRMT1 modulates MSX1 phase separation by methylating its IDR residues R150 and R157.
From: PRMT1-methylated MSX1 phase separates to control palate development
A MSX1 PTM prediction. B Representative western blot images of aDMA-MSX1 levels in HEK293T transfected with indicated plasmids. C In vitro methylation assay of MSX1-FL/R150S/R157S-mEGFP purified proteins. D–G MSX1-FL-mEGFP, MSX1-R150S-mEGFP and MSX1-R157S-mEGFP phase separation in vivo. D Representative confocal images of condensates in HEK293T. Scale bars, 10 μm. E Circularity quantification of condensates in HEK293T. MSX1-FL: n = 83, MSX1-150S: n = 187, MSX1-157S: n = 128 condensates. F FRAP analysis of MSX1 condensates in HEK293T. Scale bars, 10 μm. G FRAP quantification of MSX1 condensates. n = 3 biologically independent experiments. H–K MSX1-FL-mEGFP, MSX1-R150S-mEGFP, and MSX1-R157S-mEGFP phase separation in vitro. H Representative confocal images of condensates formed by purified proteins. Scale bars, 10 μm. I Circularity quantification of condensates formed by purified proteins. FL: n = 873, R150S: n = 1602, R157S: n = 773 condensates. J Representative images of FRAP assays assessing time-dependent fluorescence recovery of condensates formed by purified proteins. Scale bars, 1 μm. K FRAP quantification of results condensates formed by purified proteins. n = 3 biologically independent experiments. L–O MSX1-FL-mEGFP, MSX1-R150S-mEGFP, MSX1-R157S-mEGFP, MSX1-R150F-mEGFP, MSX1-R157F-mEGFP, MSX1-R150K-mEGFP and MSX1-R157K-mEGFP phase separation in vivo and in vitro. L Representative confocal images of HEPM expressing MSX1-FL-mEGFP or its mutants. Scale bars, 10 μm. M Quantitative analysis of the area of MSX1-mEGFP puncta per nucleus. n = 5 fields. N Representative DIC images of condensates formed by purified proteins. Scale bars, 10 μm. O Quantification of condensate turbidity using purified proteins. n = 3 biologically independent experiments. P, Q MSX1-FL-mEGFP, MSX1-R150S-mEGFP, MSX1-R150S-mEGFP+PRMT1, MSX1-R157S-mEGFP and MSX1-R157S-mEGFP+PRMT1 phase separation in vivo. P Representative confocal images in HEK293T. Scale bars, 10 μm. Q Circularity quantification of condensates formed in HEK293T. FL: n = 44, R150S: n = 33, R150S + PRMT1: n = 43, R157S: n = 39, R157S + PRMT1: n = 36 condensates. R, S unmethylated or methylated MSX1-FL-mEGFP, MSX1-R150S-mEGFP, and MSX1-R157S-mEGFP phase separation in vitro. R Representative DIC and fluorescence images of condensates formed by purified proteins. Scale bars, 10 μm. S Quantification of condensate sphericity using purified proteins. unmeFL: n = 25, meFL: n = 25, unmeR150S: n = 27, meR150S: n = 23, unmeR157S: n = 20, meR157S: n = 20 condensates. All data in this figure are represented as mean ± SD from at least three biologically independent experiments with similar results. Two-tailed Student’s t-test for (M, O), One-way ANOVA with Dunnett’s multiple comparisons test for (E, G, I, K), One-way ANOVA with Turkey’s multiple comparisons test for (Q, S). Source data are provided as a Source Data file.
