Oxidative phosphorylation (OXPHOS) is a vital process for energy generation, and is carried out by complexes within the mitochondria. OXPHOS complexes pose a unique challenge for cells because their subunits are encoded on both the nuclear and the mitochondrial genomes. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to mitochondria, so the co-regulation of OXPHOS genes remains largely unexplored. Here we monitor mitochondrial and nuclear gene expression in Saccharomyces cerevisiae during mitochondrial biogenesis, when OXPHOS complexes are synthesized. We show that nuclear- and mitochondrial-encoded OXPHOS transcript levels do not increase concordantly. Instead, mitochondrial and cytosolic translation are rapidly, dynamically and synchronously regulated. Furthermore, cytosolic translation processes control mitochondrial translation unidirectionally. Thus, the nuclear genome coordinates mitochondrial and cytosolic translation to orchestrate the timely synthesis of OXPHOS complexes, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for studies of global gene regulation in mitochondria.
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Balanced mitochondrial and cytosolic translatomes underlie the biogenesis of human respiratory complexes
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We thank F. Winston, T. Fox, G. Brar and members of the Churchman lab for advice and discussions; members of the O’Shea, Novina, and Springer labs for use of equipment and advice; and M. Hickman and D. Botstein for sharing the HAP1+ strain. Research supported by a Damon Runyon Cancer Research Foundation Frey Award (to L.S.C.), a Burroughs Wellcome Fund Career Award at the Scientific Interface (to L.S.C.), an Ellison Medical Foundation New Scholar in Aging Award (to L.S.C.), the National Institutes of Health F32 (to M.T.C.), and a Boehringer Ingelheim Fonds PhD Fellowship (to G.S.).
The authors declare no competing financial interests.
Reviewer Information Nature thanks P. Van Damme and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Western blot analysis of mitochondrial (Cob, Cox1, Cox2) and nuclear (Cox4) OXPHOS proteins compared to Flag-tagged mitoribosome small subunit protein MrpS17. Gapdh was used as a loading control. For gel source data, see Supplementary Fig. 1.
a–c, RNA levels (reads per kb) normalized to spike-in controls and plotted as fold changes compared to levels in log phase glucose growth for all nuclear-encoded structural components of the complexes shown (a); intron-encoded maturases (b); and nuclear and mitochondrial-encoded mitoribosome subunits (c). To calculate values for maturase transcripts, only reads not overlapping the main ORF (COX1 or COB) were considered. Group II intron splicing intermediates stably accumulate and may not represent translation-competent transcripts.
a, Serial dilution spot tests verifying tagged mitoribosome subunits are functional as they support respiratory growth on glycerol (YPG). ρ0 is a strain without mitochondrial DNA. b, Frequency of petite colonies in our corrected S288c strain (see Methods) after growth for 5 days on 0.1% glucose and 3% glycerol. BY4742 is S288c background with designer auxotrophies. Σ1278b is a strain with wild-type HAP1, and a high-fidelity allele of MIP1, MIP1[Σ], along with other differences compared to S288c. Error bars show variation due to counting, with 175–750 colonies counted for each sample. c, Lysis and immunoprecipitation buffer conditions affect mitoribosome subunit association and thus footprint retention. Left: silver staining after immunoprecipitation of the large subunit (LSU) with Mrp20–Flag and of the small subunit (SSU) with Mrps17–Flag in condition 1 (20 mM Tris pH 8.0, 200 mM KCl, 5 mM MgCl2, 0.5% lauryl maltoside), and in condition 2 (10 mM Tris pH 8.0, 50 mM NH4Cl, 10 mM MgCl2, 0.5% lauryl maltoside). Arrowheads indicate bands that appear in both immunoprecipitations in condition 2 that can be assigned to the LSU or SSU by comparison to condition 1. Asterisks mark the expected mobility of the tagged proteins. Right: northern blotting of the co-purifying RNA in each condition. For gel source data, see Supplementary Fig. 1. d, Western blot showing fractions from immunoprecipitation using optimized buffer conditions. Flag immunoprecipitation targeting the mitoribosome SSU co-purifies a haemagglutinin (HA)-tagged LSU protein. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 4 Mitoribosome profiling is robust, reproducible, and does not require translation inhibitors.
a, Mapping statistics for representative mitoribosome and cytoribosome profiling libraries from log phase glycerol-grown cells. b, Fraction of reads mapping to each frame of mitochondrial ORFs (left) and nuclear ORFs (right) in mitoribosome profiling and cytoribosome profiling data, respectively. RNA-seq reads in the left panel were treated identically to footprint reads, including size selection for library generation. c, Reproducibility between biological replicates. Each dot corresponds to the number of reads mapped to a particular position on mRNA (RPM, left), or summed number of reads mapped across each mRNA then normalized to length (RPKM, right). d, Reproducibility with and without translation inhibitors thiamphenicol (50 μg ml−1) and GTP analogue GMPPNP (1 mM).
Extended Data Figure 5 Mitochondrial and cytosolic protein synthesis on OXPHOS mRNAs is rapidly regulated.
a, b, Fold changes in relative protein synthesis (footprint RPKM values) compared to log phase glucose growth for the OXPHOS subunits synthesized in mitochondria (a; values are means of two experiments) and cytosol (b). Asterisks on heat maps indicate the subunits shown in the line plots.
Fold change data identical to those shown in Fig. 3a, but including range bars for two experiments performed from independent cultures on different days (left), and fold change translation efficiency data plotted as a scatter with the Pearson correlation coefficient (right). Dotted lines mark twofold difference. RNA-seq data used in calculating translation efficiency are from a single experiment.
Polysome profiles from samples used for cytoribosome profiling, but without RNase I treatment. Gradients were loaded with lysate from equal cell numbers, allowing overall ribosome abundances to be compared between samples. Doubling time during log phase is ~1.2 h in glucose and ~3.7 h in glycerol.
a, FACS analysis of yeast cultures treated with CCCP. Wild-type cultures were grown in glucose to mid-log phase and treated with 40 μM CCCP for the indicated times. Mitochondrial membrane potential (ΔΨm) was assessed using 1 μM tetramethylrhodamine (TMRM), which is taken up by only a fraction of the cell population (17.9% in this experiment). TMRM accumulates inside negatively charged mitochondria, producing increased fluorescence intensity (102). Loss of membrane potential dissipates probe, measured as loss of high-intensity fluorescence. b, Representative northern blots for data in Fig. 4b, c and panel e. For quantification, northern signals were normalized by relative mitoribosome recovery measured by Mrps17–Flag signal in western blots. For gel source data, see Supplementary Fig. 1. c, Northern blotting of total RNA for the indicated transcripts. For gel source data, see Supplementary Fig. 1. d, Quantification of viability assay. Cells were grown in YPD (Glu) or YPG (Gly) with or without drug for the time indicated. Cells were washed out of drug and plated on YPD for calculation of colony-forming units. CCCP (40 μM); CHX (100 μg ml−1); pentamidine (Pent; 10 μM). e, Mitochondrial translation response to inhibition of mitochondrial import with CCCP, measured by northern blotting for footprints (see b). f, Fold change in synthesis measured by cytoribosome profiling of nuclear-encoded mitochondrial mRNA-specific translational activators (colour-coded by mRNA target). For each mitochondrial mRNA, the names of the known translational activators are listed.
Extended Data Figure 9 Cytosolic OXPHOS translation response is independent of mitochondrial gene expression.
a, Metabolic labelling to measure mitochondrial translation (Mito Tln), detectable only in the presence of CHX, and cytosolic translation (Cyto Tln). Samples generated in the absence of CHX were diluted 15-fold before loading the gel compared to samples generated with CHX. Mitochondrial translation products are labelled. b, c, Full data set for experiment presented in Fig. 4d, e, showing fold changes in translation efficiency of all nuclear-encoded complex III, complex IV, and ATP synthase subunits measured by cytoribosome profiling without (−Pent) or with (+Pent) inhibition of mitochondrial translation (b) and in ρ0 cells (c), which have neither mitochondrial translation nor functional OXPHOS complexes.
a, Spot tests verifying that the ρ0 strain generated by overnight growth in ethidium bromide (see Methods) cannot respire (no growth on YPG). b, PCR (left) and qPCR (right) verifying loss of mitochondrial-encoded genes COX1, COX3, and the 21S mitochondrial rRNA gene. MRPS17 is nuclear-encoded. Bars show s.e.m. for technical triplicates.
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Couvillion, M., Soto, I., Shipkovenska, G. et al. Synchronized mitochondrial and cytosolic translation programs. Nature 533, 499–503 (2016). https://doi.org/10.1038/nature18015
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