Decoding Mitochondrial Heteroplasmy: Illuminating Regulatory Pathways via Rpn11, Mocs3, and Beyond

Abstract

Mitochondrial DNA (mtDNA) mutations underlie a diverse range of diseases that affect about one in every 5,000 individuals. These mutations often coexist alongside wildtype genomes within the same cell/organism, competing to dominant the mtDNA pool during development and aging. Various selective forces - such as purifying selection and selfish drive - can shift the abundance of these mutant genomes and thus influence the severity of mtDNA-associated disorders. The nuclear genome, which encodes more than 90% of mitochondrial proteins, can impact mtDNA transmission. How it plays out in an organism, however, remain poorly understood. In this study, I performed RNAi and compounds screens to identify nuclear-encoded proteins that regulate mtDNA competition. By examining 3,000 genes in cultured Drosophila cells stably transmitting a detrimental mtDNA mutation and quantify heteroplasmy levels via qPCR, I found that knockdown of ~1% of them can alter the heteroplasmy level during mitotic divisions. Among these candidates, MOCS3, a ubiquitin-pathway-like E1 activating enzyme, and RPN11, a subunit of the 26S proteasome complex, showed consistent effects in different cell lines. I then investigated the in vivo effects of Mocs3 and Rpn11 and discovered that removing one genomic copy of these two genes significantly reduces the functional mtDNA levels in both somatic and germline tissues of Drosophila. Several other proteasome subunits, such as RPT1, RPT2 and RPN7 etc, as well as downstream factors of MOCS3, such as AOX2, Xanthine dehydrogenase, GILT1 and ARP1 etc, also demonstrated decrease in functional mtDNA upon knockdown. Further analysis of Rpn11 knockdown clones in follicle cells within Drosophila egg chambers revealed alterations in mitophagy levels, suggesting that RPN11 may influence mitophagy to regulate mtDNA populations. In addition, I identified a compound, N, from the small molecule screen as potential treatment for mtDNA-related diseases. N treatment increased functional mtDNA levels both in vivo and in vitro in Drosophila heteroplasmy model; Patient-derived MELAS heteroplasmy fibroblast also showed decreased mutant mtDNA level upon N treatment, which guarantees further investigations of this molecules in treating mtDNA mutation related diseases. Collectively, these findings begin to unravel the complex genetic factors that influence mtDNA transmission and offer new perspectives on developing novel treatments for mitochondria diseases.