Silencing mitochondrial gene expression in living cells

Mitochondria fullfill central functions in cellular metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, central mechanisms underlying mitochondrial gene expression remain enigmatic. A lack of suitable technologies to target mitochondrial protein synthesis in living cells has limited experimental access. Here, we silence the translation of specific mitochondrial mRNAs in living cells by delivering synthetic peptide-morpholino chimeras. This approach allows comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and represents an innovative strategy to address mitochondrial gene expression in living cells. The tools that we present can readily be applied to analyze the mechanisms and pathophysiology of mitochondrial gene expression in a broad range of cellular model systems. Overall design: HEK293T cells were treated for 48 hours with chimeras targeting different mtDNA-encoded mRNAs. After discarding the media, cells were harvested in PBS, and resuspended in Trizol reagent. The RNA was purified using RNA Clean & Concentrator kit (Zymo Research) following the manufacturer's instructions. RNA quality control was assessed in a Fragment Analyzer. The NovaSeq X Plus sequencing platform (Illumina, USA) was used to perform 50 bp single-end sequencing on the samples with 9 G raw data per sample.