Mager-Heckel Anne-Marie, Entelis Nina, Brandina Irina, Kamenski Petr, Krasheninnikov Igor A, Martin Robert P, Tarassov Ivan
FRE 2375 CNRS MLPH, IPCB, Strasbourg, France.
Methods Mol Biol. 2007;372:235-53. doi: 10.1007/978-1-59745-365-3_17.
Ribonucleic acid (RNA) import into mitochondria occurs in a variety of organisms. In mammalian cells, several small RNAs are imported in a natural manner; transfer RNAs (tRNAs) can be imported in an artificial way, following expression of corresponding genes from another organism (yeast) in the nucleus. We describe how to establish and to analyze such import mechanisms in cultured human cells. In detail, we describe (1) the construction of plasmids expressing importable yeast tRNA derivatives in human cells, (2) the procedure of transfection of either immortalized cybrid cell lines or primary patient's fibroblasts and downregulation of tRNA expression directed by small interfering RNA (siRNA) as a way to demonstrate the effect of import in vivo, (3) the methods of mitochondrial RNA isolation from the transfectants, and (4) approaches for quantification of RNA mitochondrial import.
核糖核酸（RNA）导入线粒体在多种生物体中都有发生。在哺乳动物细胞中，几种小RNA以自然方式导入；转运RNA（tRNA）可通过在细胞核中表达来自另一种生物体（酵母）的相应基因后，以人工方式导入。我们描述了如何在培养的人类细胞中建立和分析这种导入机制。详细来说，我们描述了（1）在人类细胞中表达可导入的酵母tRNA衍生物的质粒构建；（2）永生化细胞杂交系或原发性患者成纤维细胞的转染程序，以及通过小干扰RNA（siRNA）下调tRNA表达以证明体内导入效果的方法；（3）从转染细胞中分离线粒体RNA的方法；（4）RNA线粒体导入定量的方法。
