Complexity and dynamics of in organello translation landscape assessed by high-resolution mitochondrial ribosome profiling

Eukaryotes house an additional protein synthesis system within the mitochondria. Given that mitochondrial translation dictates OXPHOS complex abundance and ATP levels in cells, exhaustive, quantitative, and high-resolution delineation of mitoribosome traversal is needed. Here, we developed a technique for high-resolution mitochondrial ribosome profiling and unveiled the tight regulation of mammalian in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the absolute numbers of mitoribosomes on a transcript and the elongation rate, initiation rate, and lifetime rounds of translation of individual transcripts. We also surveyed the impacts of modifications at anticodon stem loop in mt-tRNAs, including all possible decorations at 34th position, by deleting the corresponding enzymes and harnessing patient-derived mtDNA A3243G cells. Moreover, retapamulin-assisted profiling and disome profiling unveiled cryptic translation initiation sites at subcognate codons and programmed mitoribosome collision sites across the mitochondrial transcriptome, respectively. Our work provides a useful resource for delineating protein synthesis within this indispensable organelle.