Abstract
Mitochondrial import of small noncoding RNA is found in a large variety of species. In mammalian cells, this pathway can be used for therapeutic purpose, to restore the mitochondrial functions affected by pathogenic mutations. Recently, we developed mitochondrial RNA vectors able to address therapeutic oligoribonucleotides into human mitochondria. Here we provide the protocol for transfection of cultured human cells with small recombinant RNA molecules and describe two approaches useful to demonstrate their import into mitochondria: (1) isolation of RNA from purified mitochondria and quantitative hybridization analysis and (2) confocal microscopy of cells transfected with fluorescently labeled RNA. These protocols can be used in combination with overexpression or downregulation of protein import factors to detect and to evaluate their influence on the mitochondrial import of various RNAs.
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Acknowledgements
This work was supported by the CNRS (Centre National de Recherche Scientifique), the University of Strasbourg, AFM (Association Française contre les Myopathies), ANR (Agence Nationale de la Recherche), FRM (Fondation pour la Recherche Médicale), LIA collaboration program (ARNmitocure), and Labex MitoCross ANR-11-LABX-0057-MITOCROSS (National Program “Investissement d’Avenir”). I.D. was supported by ARCUS/Suprachem collaboration program; Y.T. was supported by FRM and AFM PhD fellowships.
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Dovydenko, I. et al. (2015). Mitochondrial Targeting of Recombinant RNA. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1265. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2288-8_16
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DOI: https://doi.org/10.1007/978-1-4939-2288-8_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2287-1
Online ISBN: 978-1-4939-2288-8
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