Figure 5. The pCharme/MATR3 interaction in cardiomyocytes sustains developmental genes expression.

(A) Pie charts showing the percentage of MATR3 targets in Charme^KO downregulated, invariant, or upregulated differentially expressed genes (DEGs). Significance of enrichment or depletion was assessed with two-sided Fisher’s exact test, shown below. (B) MATR3 targets (%) in the GO categories enriching Charme^KO downregulated DEGs (Down-DEGs) (see also Figure 2A). (C) Profile heatmaps of differential MATR3 CLIP-seq peaks (Charme^WT vs. Charme^KO). Normalized mean read counts of both IP samples are shown only for significant (false discovery rate [FDR] < 0.05) ‘Gain’ and ‘Loss’ peaks. (D) Upper panel: histogram showing the distribution (%) of ‘Gain’ and ‘Loss’ MATR3 peaks in pCharme DEGs. Significance of enrichment was assessed with two-sided Fisher’s exact test. Lower panel: distribution of the subset (13 out of 20) of Down-DEGs with Loss peaks in the first three GO categories identified for downregulated genes (see also Figure 2A). (E) Left panel: schematic representation of primary cells extraction from Charme^WT hearts. Once isolated, cells were plated and transfected with the specific siRNA (si-Matr3) or control siRNA (si-SCR). See ‘Materials and methods’ for details. Right panel: RT-qPCR quantification of Matr3, Cacna1c, Notch3, Myo18b, and Rbm20 RNA levels in primary cardiac cells treated with si-SCR or si-Matr3. Data were normalized to Gapdh mRNA and represent mean ± SEM of (n=4) independent biological experiments. Data information: *p<0.05; **p<0.01; ***p<0.001, unpaired Student’s t-test.

Figure 5—figure supplement 1. Study of MATR3 binding properties from MATR3 CLIP-seq Charme^WT and Charme^KO datasets.

(A) Empirical cumulative distribution functions (ECDF) showing the RNA abundance of MATR3 targets in Charme^KO condition compared to the other expressed genes. Significance was determined using a two-sided Kolmogorov–Smirnov (KS) test. (B) Principal component analysis (PCA) plot of Charme^WT and Charme^KO MATR3 CLIP-seq peaks. Plot was obtained from DiffBind package. See ‘Materials and methods’ for details. (C) Heatmap visualization of Charme^WT and Charme^KO MATR3 CLIP-seq peaks. Plot was produced by DiffBind package. Z-score of normalized reads counts is shown. See ‘Materials and methods’ for details. (D) Positional enrichment analysis of MATR3 motif in differential MATR3 CLIP-seq peaks (Charme^WT vs. Charme^KO). For each analyzed position close to peak summit, line plot displays the median CLIP-seq signal (FPM, IP1 + IP2) while heatmap displays the log₂ odds ratio of UUCUU motif enrichment. Significant enrichments (Bonferroni corrected p-value<0.05) are shown in red (seeee ‘Materials and methods’ for further details). CLIP-seq signal in Charme^KO and Charme^WT conditions is displayed for ‘Gain’ and ‘Loss’ peaks, respectively.