Mitochondrial Metabolism in the Spotlight: Maintaining Balanced RNAP III Activity Ensures Cellular Homeostasis.

RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased RNAP III activity in the S. cerevisiae maf1Δ strain is lethal when grown on a non-fermentable carbon source. This lethal phenotype is suppressed by reducing tRNA synthesis. Neither the cause of the lack of growth nor the underlying molecular mechanism have been deciphered, and this area has been awaiting scientific explanation for a decade. Our previous proteomics data suggested mitochondrial dysfunction in the strain. Using model mutant strains maf1Δ (with increased tRNA abundance) and rpc128-1007 (with reduced tRNA abundance), we collected data showing major changes in the TCA cycle metabolism of the mutants that explain the phenotypic observations. Based on 13C flux data and analysis of TCA enzyme activities, the present study identifies the flux constraints in the mitochondrial metabolic network. The lack of growth is associated with a decrease in TCA cycle activity and downregulation of the flux towards glutamate, aspartate and phosphoenolpyruvate (PEP), the metabolic intermediate feeding the gluconeogenic pathway. rpc128-1007, the strain that is unable to increase tRNA synthesis due to a mutation in the C128 subunit, has increased TCA cycle activity under non-fermentable conditions. To summarize, cells with non-optimal activity of RNAP III undergo substantial adaptation to a new metabolic state, which makes them vulnerable under specific growth conditions. Our results strongly suggest that balanced, non-coding RNA synthesis that is coupled to glucose signaling is a fundamental requirement to sustain a cell's intracellular homeostasis and flexibility under changing growth conditions. The presented results provide insight into the possible role of RNAP III in the mitochondrial metabolism of other cell types.

Glycolytic flux in Saccharomyces cerevisiae is dependent on RNA polymerase III and its negative regulator Maf1.

Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III)-driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose-rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae In a mutant compromised in RNAP III activity, there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.