Monitoring mammalian mitochondrial translation with MitoRiboSeq

Sophia Hsin Jung Li, Michel Nofal, Lance R. Parsons, Joshua D. Rabinowitz, Zemer Gitai

Research output: Contribution to journalReview articlepeer-review

Abstract

Several essential components of the electron transport chain, the major producer of ATP in mammalian cells, are encoded in the mitochondrial genome. These 13 proteins are translated within mitochondria by ‘mitoribosomes’. Defective mitochondrial translation underlies multiple inborn errors of metabolism and has been implicated in pathologies such as aging, metabolic syndrome and cancer. Here, we provide a detailed ribosome profiling protocol optimized to interrogate mitochondrial translation in mammalian cells (MitoRiboSeq), wherein mitoribosome footprints are generated with micrococcal nuclease and mitoribosomes are separated from cytosolic ribosomes and other RNAs by ultracentrifugation in a single straightforward step. We highlight critical steps during library preparation and provide a step-by-step guide to data analysis accompanied by open-source bioinformatic code. Our method outputs mitoribosome footprints at single-codon resolution. Codons with high footprint densities are sites of mitoribosome stalling. We recently applied this approach to demonstrate that defects in mitochondrial serine catabolism or in mitochondrial tRNA methylation cause stalling of mitoribosomes at specific codons. Our method can be applied to study basic mitochondrial biology or to characterize abnormalities in mitochondrial translation in patients with mitochondrial disorders.

Original languageEnglish (US)
Pages (from-to)2802-2825
Number of pages24
JournalNature Protocols
Volume16
Issue number6
DOIs
StatePublished - Jun 2021

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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