Dovydenko Ilya, Heckel Anne-Marie, Tonin Yann, Gowher Ali, Venyaminova Alya, Tarassov Ivan, Entelis Nina
UMR 7156 Genetique Moleculaire, Genomique, Microbiologie (GMGM), University of Strasbourg-CNRS, 21 Rue René Descartes, 67084, Strasbourg, France.
Methods Mol Biol. 2015;1265:209-25. doi: 10.1007/978-1-4939-2288-8_16.
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.
小非编码RNA的线粒体导入在多种物种中都有发现。在哺乳动物细胞中，该途径可用于治疗目的，以恢复受致病突变影响的线粒体功能。最近，我们开发了能够将治疗性寡核糖核苷酸导入人线粒体的线粒体RNA载体。在此，我们提供了用小重组RNA分子转染培养的人细胞的方案，并描述了两种有助于证明其导入线粒体的方法：（1）从纯化的线粒体中分离RNA并进行定量杂交分析，以及（2）对用荧光标记RNA转染的细胞进行共聚焦显微镜观察。这些方案可与蛋白质导入因子的过表达或下调相结合，以检测和评估它们对各种RNA线粒体导入的影响。
