Monitoring mammalian mitochondrial translation with MitoRiboSeq.

TitleMonitoring mammalian mitochondrial translation with MitoRiboSeq.
Publication TypeJournal Article
Year of Publication2021
AuthorsLi, SHsin-Jung, Nofal, M, Parsons, LR, Rabinowitz, JD, Gitai, Z
JournalNat Protoc
Volume16
Issue6
Pagination2802-2825
Date Published2021 06
ISSN1750-2799
KeywordsGene Expression Profiling, HCT116 Cells, Humans, Mitochondrial Ribosomes, Protein Biosynthesis, Sequence Analysis
Abstract

<p>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.</p>

DOI10.1038/s41596-021-00517-1
Alternate JournalNat Protoc
PubMed ID33953394
Grant ListDP1 AI124669 / AI / NIAID NIH HHS / United States
DP1AI124669 / / U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH) /
DP1DK113643 / / U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH) /
SU2CAACR-DT-20-16 / / EIF | Stand Up To Cancer (SU2C) /
R01 CA163591 / CA / NCI NIH HHS / United States