RNA interference (RNAi) has been thought to be a gene-silencing pathway present in most eukaryotic cells to safeguard the genome against retrotransposition. Small interfering RNAs (siRNAs) have also become a powerful tool for studying gene functions. Given the endosymbiotic hypothesis that mitochondria originated from prokaryotes, mitochondria have been generally assumed to lack active RNAi; however, certain bacteria have Argonaute homologs and various reports suggest the presence of specific microRNAs and nuclear genome (nDNA)-encoded Ago2 in the mitochondria. Here we report that transfected siRNAs are not only able to enter the matrix of mitochondria, but also function there to specifically silence targeted mitochondrial transcripts. The mitoRNAi effect is readily detectable at the mRNA level, but only recordable on relatively unstable proteins, such as the mtDNA-encoded complex IV subunits. We also apply mitoRNAi to directly determine the postulated crosstalk between individual respiratory chain complexes, and our result suggests that the controversial observations previously made in patient-derived cells might result from differential adaptation in different cell lines. Our findings bring a new tool to study mitochondrial biology.

RNA 干扰 (RNAi) 被认为是存在于大多数真核细胞中的基因沉默途径，以保护基因组免受逆转录转座。小干扰 RNA (siRNA) 也已成为研究基因功能的有力工具。鉴于线粒体起源于原核生物的内共生假说，人们普遍认为线粒体缺乏活性 RNAi。然而，某些细菌具有 Argonaute 同源物，各种报告表明线粒体中存在特定的 microRNA 和核基因组 (nDNA) 编码的 Ago2。在这里，我们报告转染的 siRNA 不仅能够进入线粒体基质，而且还在那里发挥作用以特异性地沉默靶向线粒体转录物。线粒体干扰效应在 mRNA 水平上很容易检测到，但只能在相对不稳定的蛋白质上记录，例如 mtDNA 编码的复杂 IV 亚基。我们还应用 mitoRNAi 直接确定单个呼吸链复合物之间假定的串扰，我们的结果表明，先前在患者来源的细胞中进行的有争议的观察可能是由于不同细胞系的差异适应造成的。我们的发现为研究线粒体生物学带来了一种新工具。