Monitoring the complexity and dynamics of mitochondrial translation

Since mitochondrial translation leads to the synthesis of the essential OXPHOS subunits, exhaustive and quantitative delineation of mitoribosome traversal is needed. Here, we developed a variety of high-resolution mitochondrial ribosome profiling derivatives and revealed the intricate regulation of mammalian mitochondrial translation. Harnessing a translation inhibitor retapamulin, our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the number of mitoribosomes on a transcript, the elongation rate, and initiation rate. We also surveyed the impacts of modifications at the anticodon stem loop in mt-tRNAs, including all possible modifications at the 34th position, in cells deleting the corresponding enzymes and derived from patients, and in mouse tissues. Moreover, a retapamulin-assisted derivative and mito-disome profiling revealed mtIF3-mediated translation initiation from internal ORFs and programmed mitoribosome collision sites across the mitochondrial transcriptome. Our work provides a useful platform for investigating protein synthesis within the energy powerhouse of the cell.