Regulators of mitonuclear balance link mitochondrial metabolism to mtDNA expression

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 requiring coordinated gene expression across organelles. 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 Complex IV. We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically impacts Complex IV biogenesis by acting at the intersection of mitochondrial lipid metabolism and protein synthesis, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 forms a complex with RCC1L, which together perform NDPK activity to maintain local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, NME6 modulates the activity of mitoribosome regulatory complexes, altering mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression. Ribosome profiling and total RNA-sequencing in control and NME6 KO K562 cells