Silencing of mitochondrial gene expression using polymorpholino chimeras

The human mitochondrial genome encodes 13 essential protein components of the oxidative phosphorylation complexes responsible for ATP production. Elucidation of mitochondrial gene expression and mitochondria–cellular communication has long been hindered by the lack of effective genetic tools. Import of nucleic acids such as short interfering RNA (siRNA) and guide RNA into the mitochondrial matrix has not been achieved yet, making both RNA interference and CRISPR–Cas9 inapplicable for targeting mitochondrial gene expression at the RNA or DNA level. To address these limitations, we have developed a mitochondrially targeted polymorpholino oligonucleotide chimera tool, enabling highly specific and efficient silencing of mitochondrial mRNAs by blocking mRNA–ribosome interactions. We used this approach to reveal mechanisms of bicistronic ATP synthase F0 complex subunit 8 (ATP8) and subunit 6 (ATP6) transcript expression: silencing ATP8 inhibits ATP6 translation, but silencing ATP6 does not affect ATP8. After hybridization with specific mRNA, the chimera tool also enabled the co-isolation and identification of RNA-binding proteins specific to each mitochondrial mRNA. In living cells, gene silencing affects oxidative phosphorylation complex assembly and function, and global analyses (proteomics and RNA sequencing) have uncovered both cytosolic and nuclear responses to defects in mitochondrial gene expression. Notably, this method enabled the identification of novel nuclear-encoded mitochondrial proteins crucial for the biogenesis of mitochondrial proteins. Researchers are now working on applying the chimera tool in primary cell models and whole organisms to study the effects of mitochondrial gene expression defects at tissue and organ levels.