Journal of IiME Volume 1 Issue 2 www.investinme.org Gene therapy for mitochondrial dysfunctions using optimized mRNA transport to the mitochondrial surface (continued) Figure 1: Enrichment at the mitochondrial surface of the nATP6 mRNA led to an efficient mitochondrial import of the corresponding protein A. The amount hybrid nATP6 mRNA was determined by RTPCR in RNA purifications obtained from free polysomes (FP) and mitochondrion-bound polysomes (MP). The distribution of the endogenous mRNAs SOD2 and ATP6 were also examined in both polysome fractions. Four independent experiments were compared, the results obtained are illustrated as bar graphs. The presence of the SOD2 MTS in the nATP6 mRNA allowed its enrichment in the MP fraction. Nevertheless, both the MTS and 3’UTR were required in the hybrid mRNA for allowing its exclusively sorting to the mitochondrial surface. B. The amount of the chimeric ATP6 protein was evaluated in six independent mitochondrial purifications subjected to Proteinase K (PK) digestion. This amount was compared to the quantity of ATPα protein insensitive to PK proteolysis. When the synthesis of ATP6 was directed by the gene in which both the MTS and 3’UTR of SOD2 were present, the amount of fully translocated ATP6 protein was not significantly different to the ATPα protein (bar graphs). (continued on page 25) Invest in ME Charity Nr 1114035 Page 24/72

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