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.
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Data availability
The raw FASTQ data files are deposited at NCBI’s Sequence Read Archive (SRA). The sequencing results used in the bioinformatics analysis are available under BioProject PRJNA590503 and SRA accession numbers SRR10491343, SRR10491342, SRR10491341 and SRR10491340.
Code availability
All related codes required for making the plots shown in this protocol are provided on the GitHub page at https://github.com/sophiahjli/MitoRiboSeq. These files are sufficient for readers to reproduce the bioinformatics results shown in this manuscript. The user can use the readme file with a step-by-step guide to set up the computation environment and to run the codes.
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Acknowledgements
We thank R. J. Morscher for his help with establishing the initial MitoRiboSeq method. We thank the Gitai lab and the Rabinowitz lab for their comments and suggestions. We thank S. Vianello for his support with the artistic illustration of the protocol workflow. This work is supported by funding to J.D.R. from the US National Institutes of Health (NIH) (R01CA163591 and DP1DK113643) and StandUp to Cancer (SU2CAACR-DT-20-16). Z.G. was supported by the NIH (DP1AI124669).
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S.H.-J.L. and M.N. developed the protocol. S.H.-J.L. and L.P. developed the bioinformatics analysis pipeline. S.H.-J.L., M.N. and Z.G. wrote the paper with help from J.D.R.
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Peer review information Nature Protocols thanks Jean-Michel Cioni, Norbert Huebner and the other, anonymous reviewer(s) for their contribution to the peer review of this work.
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Morscher, R. et al. Nature 554, 128–132 (2018): https://doi.org/10.1038/nature25460
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Li, S.HJ., Nofal, M., Parsons, L.R. et al. Monitoring mammalian mitochondrial translation with MitoRiboSeq. Nat Protoc 16, 2802–2825 (2021). https://doi.org/10.1038/s41596-021-00517-1
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DOI: https://doi.org/10.1038/s41596-021-00517-1
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