Laboratory of Systems and Synthetic Biology, Chair of Drugs and Cosmetics Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland.
Certara UK Limited, Sheffield S1 2BJ, UK.
Int J Mol Sci. 2023 Sep 29;24(19):14763. doi: 10.3390/ijms241914763.
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 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 (with increased tRNA abundance) and (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 C 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. , 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.
RNA 聚合酶 III（RNAP III）全酶活性及其产物的加工与低等和高等真核生物的几种代谢功能障碍有关。RNAP III 驱动的非编码 RNA 合成活性的改变会导致葡萄糖代谢的广泛变化。在非可发酵碳源上生长时， 菌株中 RNAP III 活性的增加是致命的。这种致死表型可通过降低 tRNA 合成来抑制。缺乏生长的原因和潜在的分子机制尚未被破解，这一领域已经等待了十年的科学解释。我们之前的蛋白质组学数据表明 菌株存在线粒体功能障碍。使用模型突变株 （tRNA 丰度增加）和 （tRNA 丰度降低），我们收集的数据显示突变体的 TCA 循环代谢发生了重大变化，这些变化解释了表型观察结果。基于 C 通量数据和 TCA 酶活性分析，本研究确定了线粒体代谢网络中的通量限制。缺乏生长与 TCA 循环活性降低以及谷氨酸、天冬氨酸和磷酸烯醇丙酮酸（PEP）通量下调有关，PEP 是糖异生途径的代谢中间产物。 菌株由于 C128 亚基的突变而无法增加 tRNA 合成，在非发酵条件下 TCA 循环活性增加。总之，RNAP III 活性非最佳的细胞会对新的代谢状态进行大量适应，这使得它们在特定的生长条件下变得脆弱。我们的结果强烈表明，与葡萄糖信号偶联的平衡、非编码 RNA 合成是维持细胞在不断变化的生长条件下的细胞内稳态和灵活性的基本要求。所呈现的结果为 RNAP III 在其他细胞类型的线粒体代谢中的可能作用提供了见解。
