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Regulators of mitonuclear balance link mitochondrial metabolism to mtDNA expression

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Cell Culture

SUBMITTER: Nicholas Kramer

LAB HEAD: Stirling Churchman

PROVIDER: PXD044812 | Pride | 2023-08-23

REPOSITORIES: Pride

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			Action	DRS
	NME6-KO-1_100576.raw	Raw
	NME6-KO-1_86337_100576.pep.xml	Pepxml
	NME6-KO-2_100577.raw	Raw
	NME6-KO-2_86368_100577.pep.xml	Pepxml
	NME6-KO-3_100578.raw	Raw

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Project description:Background: Transcription control of mitochondrial metabolism is essential for cellular function. A better understanding of this process will aid the elucidation of mitochondrial disorders, in particular of the many genetically unsolved cases of oxidative phosphorylation (OXPHOS) deficiency. Yet, to date only few studies have investigated nuclear gene regulation in the context of OXPHOS deficiency. In this study, we combined RNA sequencing of human complex I-deficient patient cells across 32 conditions of perturbed mitochondrial metabolism, with a comprehensive analysis of gene expression patterns, co-expression calculations and transcription factor binding sites. Results: Our analysis shows that OXPHOS genes have a significantly higher co-expression with each other than with other genes, including mitochondrial genes. We found no evidence for complex-specific mRNA expression regulation in the tested cell types and conditions: subunits of different OXPHOS complexes are similarly (co-)expressed and regulated by a common set of transcription factors. However, we did observe significant differences between the expression of OXPHOS complex subunits compared to assembly factors, suggesting divergent transcription programs. Furthermore, complex I co-expression calculations identified 684 genes with a likely role in OXPHOS biogenesis and function. Analysis of evolutionarily conserved transcription factor binding sites in the promoters of these genes revealed almost all known OXPHOS regulators (including GABP, NRF1/2, SP1, YY1, E-box factors) and a set of six yet uncharacterized candidate transcription factors (ELK1, KLF7, SP4, EHF, ZNF143, and EL2). Conclusions: OXPHOS genes share an expression program distinct from other mitochondrial genes, indicative of targeted regulation of this mitochondrial sub-process. Within the subset of OXPHOS genes we established a difference in expression between subunits and assembly factors. Most transcription regulators of genes that co-express with complex I are well-established factors for OXPHOS biogenesis. For the remaining six factors we here suggest for the first time a link with transcription regulation in OXPHOS deficiency. RNA-SEQ of whole cell RNA in 2 control and 2 complex I deficient patient fibroblast cell lines treated with 4 compounds in duplicate, resulting in a total of 2x2x4x2=32 samples

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Regulators of mitonuclear balance link mitochondrial metabolism to mtDNA expression

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