The effect of loss of RTN4IP1 on mitochondrial function. (360G-Wellcome-211645_Z_18_Z)

Mitochondria are complex cellular organelles that harness the process of oxidative phosphorylation (OXPHOS) to provide cellular energy, but defects in this process can cause mitochondrial diseases that are associated with a wide range of clinical symptoms, severity and age of onset. Recently, whole-exome sequencing identified pathogenic variants in RTN4IP1 in patients presenting with mitochondrial disorders. RTN4IP1 patient derived cells showed a complete loss of this protein, a mitochondrial respiratory chain Complex I assembly defect and reduction in mitoribosome formation. This project will utilise patient-derived fibroblasts harbouring pathogenic variants in the RTN4IP1 gene and the CRISPR/Cas9-mediated RTN4IP1 knockout cell lines to determine the precise role of this protein in mitochondrial function. First, we will examine cellular growth of RTN4IP1 patient and CRISPR/Cas9 knockout cell lines in galactose media forcing the utilisation of the OXPHOS system. Then, RTN4IP1 localisation within the organelle will be determined by mitochondrial sub-fractionation and Western blotting. In addition, the steady-state levels of OXPHOS and mitoribosomal proteins will be assessed in RTN4IP1-deficient cells. Finally, sucrose density gradient centrifugation will be used to assess a potential role of RTN4IP1 in the mitoribosome biogenesis. Key words: mitochondrial disease, RTN4IP1, OXPHOS, mitoribosome