Background Oxidative phosphorylation (OXPHOS) complexes consist of nuclear and mitochondrial DNA-encoded subunits. Their biogenesis requires cross-compartment gene regulation to mitigate the accumulation of disproportionate subunits. To determine how human cells coordinate mitochondrial and nuclear gene expression processes, we tailored ribosome profiling for the unique features of the human mitoribosome. Results We resolve features of mitochondrial translation initiation and identify a small ORF in the 3′ UTR of MT-ND5. Analysis of ribosome footprints in five cell types reveals that average mitochondrial synthesis levels correspond precisely to cytosolic levels across OXPHOS complexes, and these average rates reflect the relative abundances of the complexes. Balanced mitochondrial and cytosolic synthesis does not rely on rapid feedback between the two translation systems, and imbalance caused by mitochondrial translation deficiency is associated with the induction of proteotoxicity pathways. Conclusions Based on our findings, we propose that human OXPHOS complexes are synthesized proportionally to each other, with mitonuclear balance relying on the regulation of OXPHOS subunit translation across cellular compartments, which may represent a proteostasis vulnerability.
Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells, in which gene expression must be coordinated across organelles using distinct pools of ribosomes. How cells produce and maintain the accurate subunit stoichiometries for these OXPHOS complexes remains largely unknown. To identify genes involved in dual-origin protein complex synthesis, we performed FACS-based genome-wide screens analyzing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of cytochrome c oxidase (Complex IV). We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically regulates Complex IV biogenesis by interacting with mitochondrial protein synthesis machinery, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS complex biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 maintains local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, through stabilizing interactions with RCC1L, NME6 modulates the activity of mitoribosome regulatory complexes, leading to disruptions in mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression. Finally, we present these screens as a resource, providing a catalog of genes involved in mitonuclear gene regulation and OXPHOS biogenesis.
Mitochondria play a critical role in cellular metabolism primarily through hosting the oxidative phosphorylation (OXPHOS) machinery that is encoded by mitochondrial DNA (mtDNA) and nuclear DNA, with each genome separately regulated in their respective compartments. To unravel how the two gene expression systems collaborate to produce the OXPHOS complexes, the regulatory principles controlling the production of mtDNA-encoded proteins need to be elucidated. Here, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to gain insight into which steps of gene expression experience the most regulatory control. Our analysis revealed a unique balance between the rapid turnover and high accumulation of mt-mRNA, leading to a 700-fold higher transcriptional output than nuclear-encoded OXPHOS genes. Additionally, we observed that mt-mRNA processing and its association with the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. These data resulted in a model of mtDNA expression that is predictive across human cell lines, revealing that differential turnover and translation efficiencies are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome, French-Canadian type, we found that disrupting the responsible nuclear-encoded gene, LRPPRC, perturbs OXPHOS biogenesis predominantly through altering mt-mRNA stability. Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle for decoding the regulatory principles of mtDNA expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.