Merck

Monitoring mammalian mitochondrial translation with MitoRiboSeq.

Nature protocols (2021-05-07)
Sophia Hsin-Jung Li, Michel Nofal, Lance R Parsons, Joshua D Rabinowitz, Zemer Gitai
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.

MATERIALS
Product Number
Brand
Product Description

Roche
Nuclease S7, Micrococcal nuclease, from Staphylococcus aureus
Roche
DNase I recombinant, RNase-free, from bovine pancreas, expressed in Pichia pastoris
Roche
cOmplete, Mini Protease Inhibitor Cocktail, Tablets provided in EASYpacks