Fig. 8

CLUH affects the subcellular distribution of some mRNAs coding for mitochondrial proteins. A Venn diagram depicting the intersection between mitochondrial CPMPs (purple) (see Fig. 4E) and the mRNAs identified by APEX-seq at the mitochondrial outer membrane (OMM) (green) [6]. Mitochondrial proteins are selected according to their presence in the Mitocarta 3.0 database. The OMM localized mRNAs correspond to OMM biotinylated mRNA in the presence of cycloheximide significantly enriched above the background (FDR < 10%) and positively enriched (log2FC >0) over non-specific cytosolic biotinylation. B Graph showing RT-qPCR analysis of mRNA enrichment in crude mitochondrial fraction from CLUH KO (orange) and WT HCT116 cells (red). The enrichment of specific mRNA (normalized to EIF5A levels) is calculated relative to the total RNA from non-fractionated input sample (% of input) and shown on log2 scaled y-axis. The box summarizes triplicate experiments, showing the upper, the lowest, and mean enrichment values. mRNAs showing the highest variation between WT and CLUH KO are highlighted by the orange shadow. C Schematic representation of the experimental workflow to extract RNA and proteins from polysomal fractions from both total and “crude” mitochondrial fractions. D Graph showing RT-qPCR analysis of mRNA enrichment in the polysomes from crude mitochondrial fraction in CLUH KO (orange) and WT HCT116 cells (red). The enrichment of specific mRNA (normalized to EIF5A levels) is calculated relative the total RNA from non-fractionated input sample (% of input) and shown on log2 scaled y-axis. The box summarizes triplicate experiments, showing the upper, the lowest, and mean enrichment values. mRNAs showing the highest variation between WT and CLUH KO are highlighted by the orange shadow