Allotopic mRNA localization to the mitochondrial surface rescues respiratory chain defects in fibroblasts harboring mitochondrial DNA mutations affecting complex I or v subunits.
ABSTRACT The possibility of synthesizing mitochondrial DNA (mtDNA)-coded proteins in the cytosolic compartment, called allotopic expression, provides an attractive option for genetic treatment of human diseases caused by mutations of the corresponding genes. However, it is now appreciated that the high hydrophobicity of proteins encoded by the mitochondrial genome represents a strong limitation on their mitochondrial import when translated in the cytosol. Recently, we optimized the allotopic expression of a recoded ATP6 gene in human cells, by forcing its mRNA to localize to the mitochondrial surface. In this study, we show that this approach leads to a long-lasting and complete rescue of mitochondrial dysfunction of fibroblasts harboring the neurogenic muscle weakness, ataxia and retinitis Pigmentosa T8993G ATP6 mutation or the Leber hereditary optic neuropathy G11778A ND4 mutation. The recoded ATP6 gene was associated with the cis-acting elements of SOD2, while the ND4 gene was associated with the cis-acting elements of COX10. Both ATP6 and ND4 gene products were efficiently translocated into the mitochondria and functional within their respective respiratory chain complexes. Indeed, the abilities to grow in galactose and to produce adenosine triphosphate (ATP) in vitro were both completely restored in fibroblasts allotopically expressing either ATP6 or ND4. Notably, in fibroblasts harboring the ATP6 mutation, allotopic expression of ATP6 led to the recovery of complex V enzymatic activity. Therefore, mRNA sorting to the mitochondrial surface represents a powerful strategy that could ultimately be applied in human therapy and become available for an array of devastating disorders caused by mtDNA mutations.
- SourceAvailable from: sld.cuNew England Journal of Medicine 07/2003; 348(26):2656-68. · 51.66 Impact Factor
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ABSTRACT: 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.RNA 08/2006; 12(7):1408-17. · 5.09 Impact Factor
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ABSTRACT: Oxidative phosphorylation, i.e., ATP synthesis by the oxygen-consuming respiratory chain (RC), supplies most organs and tissues with a readily usable energy source, being functional before birth. Consequently, RC deficiencies can theoretically give rise to any symptom, in any organ or tissue, at any age and with any mode of inheritance, because of the twofold genetic origin of RC components (nuclear DNA and mitochondrial DNA). It was long wrongly considered that RC disorders originate from mutations of mitochondrial DNA, because for a long time only mutations or deletions of mitochondrial DNA were identified. However, the number of known disease-causing mutations in nuclear genes is steadily growing. These genes encode the various subunits of each complex, ancillary proteins functioning at different stages of holoenzyme biogenesis, including transcription, translation, chaperoning, addition of prosthetic groups, and protein assembly, and various enzymes involved in mitochondrial DNA metabolism.Journal of the American Society of Nephrology 01/2004; 14(12):2995-3007. · 8.99 Impact Factor