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. 2023 Jul 18;14:4312. doi: 10.1038/s41467-023-39965-6

Fig. 4. Mislocalization of RS-domain RBM20 mutants is caused by loss of interaction with TNPO3.

Fig. 4

a ICS-measured DRAQ5:RBM20 correlation in WT or P633L iPSC-CMs transfected with control (Ctr) or TNPO3 siRNA. b DAPI:RBM20 correlation (based on Supplementary Fig. 7a). Each dot represents a Pearson coefficient for at least five cells, n = 3. c qPCR analysis of TTN exon 242 splicing-out in WT or P633L iPSC-CMs with Ctr or TNPO3 siRNA normalized to GAPDH (mean fold change versus the RBM20-WT with control siRNA, with standard errors, two biological replicates with two technical replicates each). d ICS-measured DRAQ5:RBM20 correlation for HeLa expressing eGFP-WT-, -P633L-, -R634Q-, or -RSS-RBM20, with Ctr or TNPO3 siRNA. e DAPI:RBM20 correlation (based on Supplementary Fig. 8a). Each dot represents a Pearson coefficient for at least five cells, n = 3. f Superimposed AlphaFold2 models of the RRM-RS domain of RBM20 (amino acid 511–673) as WT (gray), P633L (light blue), or R634Q (salmon). g Representative AlphaFold2 model of RBM20’s WT RRM-RS domain (blue, amino acid 511–673) in complex with TNPO3 (gray, full-length, amino acid 1-923). The PRSRSP stretch (amino acid 633–638) in RBM20 is highlighted as red spheres. h Predicted changes in binding affinity between TNPO3 and RBM20 upon RS-domain mutations. N = 20 AlphaFold models of the wild-type RBM20-TNPO3 complex were used to calculate the affinity change with a point mutation (see “Methods”). i Quantification of TNPO3 co-immunoprecipitating with RBM20 based on western blot analysis, representative image is displayed in (j) (n = 3, means with standard errors). j Western blot analysis of RBM20, TNPO3, and MOV10 in the cytoplasmic fraction of HeLa, and their co-immunoprecipitation with eGFP-RBM20 (Neg = negative no-bait control). k Quantification of TNPO3 peptides identified by mass spectrometry in the cytoplasmic fractions of co-immunoprecipitants with indicated cells, normalized to Neg, n = 3. Boxplots (b, e, h, k) display quartiles Q1, Q2 (center), and Q3, with whiskers extending to the furthest data point within 1.5 times the IQR. Ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t-test for (b), and one-way ANOVA with Tukey’s HSD post test for (c), (e), all two-tailored. Actual P values are shown in the source data file.