Fig. 3: Genome-wide ICS screen identifies TNPO3 as the main nuclear transporter of RBM20. | Nature Communications

Fig. 3: Genome-wide ICS screen identifies TNPO3 as the main nuclear transporter of RBM20.

From: Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3

Fig. 3

a Schematic outline of the ICS screen. Six genome-wide libraries were applied to HeLa cells expressing eGFP-RBM20-WT and Tet::Cas9, with 100 cells per gRNA coverage. Cells were sorted based on the correlation between RBM20 and DRAQ5 into 7% higher and 7% lower fractions at final coverage of 500 cells per gRNA per sorted bin. Unsorted input samples were collected too. b Reads were combined in silico to one dataset, and hits were called using MAUDE69. Genes are ranked by their statistical significance. The horizontal dashed lines indicate an FDR of 1%. Positive/negative regulators with FDR < 1% are marked in red and blue, respectively. c Scatter plot of fold changes visualizing gRNA abundance changes in higher (x axis) and lower (y axis) sorted bins compared with the plasmid library. Red and blue dots indicate statistically significant positive and negative regulators, respectively (FDR < 5% according to MAUDE). Labeled are positive regulators selected for future analyses. d The impact of single knockouts of the selected hits (one gRNA per gene picked based on the strongest Z-score from the pooled screen) on RBM20 localization tested with ICS. The top row in the heatmap shows the log10(FDR) value for each candidate from the screen. The phenotype in the second row represents the standardized difference in RBM20 localization between the knockout (KO) and control cell populations (log2 of the ratio between cell fraction with Pearson coefficient (DRAQ5:RBM20) > 0.7 in the KO divided by cell fraction with Pearson coefficient (DRAQ5:RBM20) >0.7 in the control). e DAPI:RBM20 correlation quantified based on fluorescence microscopy analysis shown in Supplementary Fig. 5g, for the single KOs indicated. Each dot represents a Pearson correlation coefficient R for at least five cells, n = 3. Boxplots 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 < 0.001, one-way ANOVA with Tukey’s HSD post test (two-sided). Actual P values are shown in the source data file.

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