Fig. 7

CLUH requires active translation to bind mRNAs coding for mitochondrial proteins and does not affect their translation efficiency. A RT-qPCR analysis of RNA immunoprecipitation (RIP) experiments performed on the endogenous CLUH protein in wild-type HCT116 (red) and CLUH KO (orange) cells. B RT-qPCR analysis of RIP experiments performed on rescued HCT116 CLUH KO cells. The cells are transduced to stably express either the tagged wildtype CLUH (CLUH_WT, red) or the tagged mutant CLUH (CLUH_∆TPR, yellow). Cells expressing a tagged GFP protein (GFP, gray) are used as background control. C RT-qPCR analysis of RIP experiment performed on wild-type HCT116 cells in translation inhibition conditions. Cells are treated with 100μg/mL of puromycin (Puro) or not (mock). A–C The CLUH associated mRNAs are enriched using CLUH-specific antibodies and measured by RT-qPCR. The enrichment of specific mRNA (normalized to GAPDH or SUB1 levels) is calculated relative to the input sample (% of input). mRNAs coding for CPMPs are highlighted by the orange shadow. The error bars correspond to the standard deviation of three independent experiments. The average value for each replicate is indicated by a dot. D Representative graphs of polysome profilings of WT and CLUH KO HCT116 cells. The y-axis corresponds to the absorbance at 260 nm and the x-axis to the distance in the sucrose gradient. The polysomal fractions used for further experiments are highlighted by the orange shadow. E RT-qPCR analysis of mRNA in polysomal fractions from the WT and CLUH KO cells. The enrichment of specific mRNA (normalized to EIF5A levels) is calculated relative to the input sample (% of input). The error bars correspond to the standard deviation of three independent experiments. The average value for each replicate is indicated by a dot. mRNAs coding for CPMPs are highlighted in orange. F Scatter plot comparing the abundance of all proteins identified by mass spectrometry in pure mitochondrial fraction (see Fig. 4F) of CLUH KO and WT HCT1116 cells. The x-axis and the y-axis show to the Z-score of the mean abundance of each protein in the WT HCT116 and CLUH KO samples, respectively. The abundance of each protein is calculated by dividing the mean spectral count of three replicate samples by the protein size. Proteins found in the Mitocarta 3.0 database are shown in red. The full dataset is available in Table S6. (G) Representative western blot analysis of polysome profiling of WT HCT116 cells. Each fraction corresponds to 3.3 mm of the sucrose gradient and fractions are numbered from 1 to 23. Total protein extracts are used as controls. Indicated proteins are revealed using specific antibodies