Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA.
Curr Genet. 2022 Dec;68(5-6):581-591. doi: 10.1007/s00294-022-01248-w. Epub 2022 Aug 3.
Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules. Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery.
许多生物过程涉及能够短暂组装成细胞功能所必需的亚细胞隔室的蛋白质。一个过程是 RNA 聚合酶 II 转录循环，它涉及起始、延伸、新生 RNA 的共转录修饰和终止。必需的酵母转录终止因子 Nab3 是终止小非编码 RNA 所必需的，并且在葡萄糖去除时积累到紧凑的核颗粒中。Nab3 核颗粒的积累在酵母菌株中具有不同的穿透性，并且更高的 Nab3 颗粒积累表型与 petite 菌株相关，表明颗粒解体可能存在 ATP 依赖性机制。在这里，我们通过药物处理或核编码 ATP 合酶亚基基因缺失来证明线粒体氧化磷酸化的解偶联足以增加 Nab3 颗粒的积累，并导致在长时间葡萄糖剥夺期间无法增殖，这需要呼吸作用。此外，通过从 petite 倾向的菌株中富集呼吸能力强的细胞，我们从相对较高的菌株中产生了低颗粒积累的菌株，为呼吸能力和 Nab3 颗粒之间提供了另一个联系。与涉及 ATP 的颗粒积累的结果一致，向半透化细胞中添加细胞外 ATP 足以减少 Nab3 颗粒的积累。删除编码激酶的 SKY1 基因，该激酶磷酸化核 SR 重复蛋白并参与有效的应激颗粒解体，也导致颗粒积累增加。这一观察结果暗示 Sky1 参与 Nab3 颗粒的生物发生。总之，这些发现表明，在终止因子颗粒组装和解组装之间通常存在由 ATP 依赖性核机制介导的平衡。
