PROJECT SUMMARY The coordinated expression of the nuclear and the mitochondrial genomes is critical for mitochondrial function; yet, what regulates this process in humans is unclear. This coordination, termed mitonuclear balance, is especially important for the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) complexes, which are composed of >90 proteins encoded by both the nuclear and the mitochondrial genomes. A significant challenge in gene regulation is the synthesis of these complexes, as the two physically separate genomes must coordinate their gene expression programs to produce the correct ratios of the subunits, ensuring proper complex assembly and function. The imbalanced production of OXPHOS subunits can be detrimental to the cell; orphan subunits and assembly intermediates lead to the accumulation of reactive oxygen species, which can lead to mitochondrial dysfunction and mitochondrial disorders, including neurodegeneration and metabolic conditions. Using a FACS-based CRISPR screen developed by our lab, we recently discovered that metabolism-related factors play a role in the balanced expression of OXPHOS complex IV subunits in human cells, thus linking certain aspects of cellular metabolism to mitonuclear balance. The overall objective of this proposal is to identify novel factors that regulate OXPHOS protein biogenesis and determine their mechanism of regulation. The central hypothesis is that diverse aspects of metabolism influence the expression of the two genomes across compartments for balanced OXPHOS protein synthesis. In Aim 1, I will investigate the function of MMADHC, a protein involved in vitamin B12 metabolism, and which is a putative regulator of complex IV subunit synthesis. I will determine the role of MMADHC on OXPHOS protein biogenesis and mitochondrial health, its impact on transcription and translation of OXPHOS subunits, and its role in establishing critical metabolite pools. In Aim 2, I will use our CRISPR/FACS screen to individually knock-out ~3000 metabolism-related genes, and identify genes whose loss leads to imbalanced expression of dual-origin subunits of complex I. I will determine the gene regulatory roles of the top hits, and whether they are regulators of complex I subunits specifically, or of OXPHOS complexes in general. I will conduct the proposed research at Harvard Medical School, in the lab of Dr. L. Stirling Churchman, who is an expert on nuclear and mitochondrial gene expression and who has developed a variety of sequencing technologies for studying the different levels of gene regulation. This proposal is part of a comprehensive professional training plan to prepare me for a career as an independent research scientist investigating coordination of gene expression across the nucleocytosolic and mitochondrial compartments, and its dysfunction in disease and development. With the completion of the proposed work, the results will uncover general pathways involved in mitonuclear bala...