mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, INSERM U592 and Université Pierre et Marie Curie (UPMC-Paris6), Hôpital St. Antoine, 75571 Paris, Cedex 12 France.
RNA (Impact Factor: 4.94). 08/2006; 12(7):1408-17. DOI: 10.1261/rna.18206
Source: PubMed


As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface-the region coding for the mitochondrial targeting sequence and the 3'UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3'UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3'UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity.

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    • "Upon proof of principle in the yeast Saccharomyces cerevisiae (Nagley et al., 1988), such allotopic expression of proteins that are normally gene products of the mtDNA was developed in mammalian model systems (Guy et al., 2002; Ojaimi et al., 2002). In further studies based on subcellular mRNA localization data (Sylvestre et al., 2003), the allotopically expressed transcripts were targeted to the mitochondrial surface, so as to promote co-translational import of the corresponding proteins into the organelles (Bonnet et al., 2008; Ellouze et al., 2008; Kaltimbacher et al., 2006). As a whole, rescue of mtDNA mutation-triggered deficient phenotype was both claimed and contradicted, leaving the issue open (Bokori-Brown and Holt, 2006; Oca-Cossio et al., 2003; Perales-Clemente et al., 2010). "
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    • "A weaker band was always detected with both anti-Flag and anti-ND1 antibodies in LHON #2 + WT-ND1 cells of about ~ 37 kDa that could correspond to the precursor form of the fusion protein (⁎). The ATPα protein had an apparent molecular weight of ~ 65 kDa as previously observed in HeLa cells [34]. Densitometric analyses were performed with signals obtained for three independent Western blots in which each protein extract was tested (Quantity One Biorad software system). "
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