Monitoring the complexity and dynamics of mitochondrial translation

Eukaryotes house the additional protein synthesis system in mitochondria. Given the mitochondrial translation dictates OXPHOS complex abundance and then ATP level in cells, the exhaustive quantitative, and high-resolution delineation of mitoribosome traversal has been awaited. Here, we developed a high-resolution mitochondrial ribosome profiling and unveiled tight regulation of in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the absolute mitoribosome numbers on a transcript, elongation rate, initiation rate, and lifetime translation round on a transcript. We also surveyed the impacts of all the 34th position modifications on mt-tRNAs, including f5C34, m5C34, tm5U34, tm5s2U34, s2U34, and Q34, as well as t6A37 by the responsible enzyme deletions. Moreover, retapamulin-assisted profiling and disome profiling unveiled cryptic translation initiation sites at sub-cognate codons and the programmed mitoribosome collision sites across the mitochondrial transcriptome, respectively. Our work provides a useful resource to delineate protein synthesis within the bioenergetic powerhouse. Overall design: Ribo-Seq, RNA-Seq, Ribo-Calibration, MitoIP-Ribo-seq, MItoIP-Thor-Ribo-Seq, MitoIP-Thor-Disome-Seq.