Transcription of human mitochondrial DNA (mtDNA) begins from specific transcription promoters. In strand-asynchronous mtDNA replication, transcripts from the light-strand promoter serve as primers for leading-strand synthesis at the origin of the H-strand replication (OH). A 7S DNA strand, a presumed aborted replication product, is also synthesized from OH. Transition from RNA synthesis to DNA synthesis at OH is crucial for balancing replication with transcription, yet the mechanism remains unclear. Herein, we examine the role of mitochondrial transcription elongation factor (TEFM) in this process. TEFM knockout results in decreased 7S DNA, strand-asynchronous replication intermediates, and mtDNA copy number, all of which are concordant with downregulation of RNA-to-DNA transition at OH. Conversely, levels of tRNAs encoded near transcription promoters increase, indicating enhanced transcription initiation frequency. Taken together, we propose that, in addition to conferring processivity to the mitochondrial RNA polymerase, TEFM plays a crucial role in maintaining the balance between mitochondrial transcription and replication.

Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

Chemical acetylation is postulated to occur in mitochondria. Mitochondrial transcription factor A (TFAM or mtTFA), a mitochondrial transcription initiation factor as well as the major mitochondrial nucleoid protein coating the entire mitochondrial genome, is proposed to be acetylated in animals and cultured cells. This study investigated the properties of human TFAM, in conjunction with the mechanism and effects of TFAM acetylation in vitro. Using highly purified recombinant human TFAM and 3 kb circular DNA as a downsized mtDNA model, we studied how the global TFAM-DNA interaction is affected/regulated by the quantitative TFAM-DNA relationship and TFAM acetylation. Results showed that the TFAM-DNA ratio strictly affects the TFAM property to unwind circular DNA in the presence of topoisomerase I. Mass spectrometry analysis showed that in vitro chemical acetylation of TFAM with acetyl-coenzyme A occurs preferentially on specific lysine residues, including those reported to be acetylated in exogenously expressed TFAM in cultured human cells, indicating that chemical acetylation plays a crucial role in TFAM acetylation in mitochondria. Intriguingly, the modification significantly decreased TFAM's DNA-unwinding ability, while its DNA-binding ability was largely unaffected. Altogether, we propose TFAM is chemically acetylated in vivo, which could change mitochondrial DNA topology, leading to copy number and gene expression modulation.

Human DNA polymerase γ (POLG) is a mitochondria-specific replicative DNA polymerase consisting of a single catalytic subunit, POLGα, and a dimeric accessory subunit, POLGβ. To gain a deeper understanding of the role of POLGβ, we knocked out this protein in cultured human cybrid cells and established numerous knockout clones. POLGβ-knockout clones presented a clear phenotype of mitochondrial DNA loss, indicating that POLGβ is necessary for mitochondrial DNA replication. Moreover, POLGβ-knockout cells showed a severe decrease in POLGα levels and acute suppression of POLGβ expression efficiently down-regulated POLGα levels. These results suggest that, in addition to its role as the processivity factor of POLG, POLGβ acts as a POLGα stabilizer, an important role for POLGβ in mitochondrial DNA maintenance.

OBJECTIVE: Hepatic steatosis, the early stage of nonalcoholic fatty liver disease (NAFLD), currently lacks targeted pharmacological treatments. G protein-coupled receptors (GPCRs) in hepatocytes differentially regulate lipid metabolism depending on their coupling profile of G protein subtypes. Unlike Gs, Gi, and Gq signaling, the role of G12 signaling in hepatic steatosis remains elusive. The objective of this study was to investigate the effect of G12 signaling on hepatic steatosis and obesity and its mechanisms. METHODS: We generated mice expressing a G12-coupled designer GPCR in a liver-specific manner. We performed phenotypic analysis in the mice under the condition of fasting (acute hepatic steatosis model) or high-fat diet feeding (chronic hepatic steatosis model). RESULTS: In acute and chronic hepatic steatosis models, chemogenetic activation of hepatic G12 signaling suppressed the progression of hepatic steatosis. The treatment led to an increased triglyceride secretion with little effect on mitochondrial respiratory activity, fatty acid oxidation, de novo lipogenesis, and fatty acid uptake. Furthermore, in a high-fat-diet-induced obesity model, activation of the G12-coupled designer GPCR exerted anti-obesity effects with increased whole-body energy expenditure and fat oxidation. Anti-FGF21 antibody treatment showed that the anti-obesity effects of the hepatic G12D activation relied in part on the hepatokine FGF21. CONCLUSIONS: Our findings indicate that the activation of G12 signaling in the liver has the potential to prevent hepatic steatosis and obesity. This discovery provides a strong rationale for the development of drugs targeting G12-coupled GPCRs expressed in the liver.