Coupling of RNA polymerase III assembly to cell cycle progression in <i>Saccharomyces cerevisiae</i>

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a <i>cold-sensitive</i> mutation, <i>rpc128-1007</i>, in the yeast gene encoding the second largest Pol III subunit, Rpc128. <i>rpc128-1007</i> is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that <i>rpc128-1007</i> mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most <i>rpc128-1007</i> mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the <i>rpc128-1007</i> mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double <i>rpc128-1007 maf1</i>Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with <i>rpc128-1007</i> is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase.

酵母与人类的RNA聚合酶组装过程，被认为发生于其核输入之前的细胞质中。我们此前的研究在酵母编码RNA聚合酶III（Pol III）第二大亚基Rpc128的基因中，发现了一处冷敏感型（cold-sensitive）突变rpc128-1007。该突变与Pol III复合物组装缺陷相关，并因此导致tRNA合成水平降低。本研究发现，rpc128-1007突变体细胞大多处于未出芽状态，且体积大于野生型细胞。流式细胞术检测结果显示，多数rpc128-1007突变体细胞的DNA含量处于G1期水平，表明该突变会引发显著的G1期细胞周期延迟。过表达编码Pol III组装/转运因子Rbs1的基因，可抵消rpc128-1007突变体中出现的G1期阻滞，并恢复突变体细胞的野生型形态。与此同时，Rbs1缺失细胞的G1期退出过程出现轻度延迟，表明Rbs1对于细胞周期的正常时序推进必不可少。我们利用tRNA合成水平得以恢复的双突变体rpc128-1007 maf1Δ，证实了rpc128-1007相关的Pol III组装缺陷是引发细胞周期阻滞的主要原因。综合以上研究结果，我们认为Pol III复合物组装受损与G1期细胞周期抑制存在关联。

搜集汇总

背景与挑战

背景概述

该数据集研究了酿酒酵母中RNA聚合酶III组装与细胞周期进程的耦合机制，通过rpc128-1007突变体实验，发现Pol III组装缺陷导致tRNA合成减少，并引起细胞在G1期停滞；同时证实Rbs1因子在此过程中起关键调节作用，恢复其表达可缓解细胞周期延迟。数据集包含补充图表文件，适用于生物物理学、细胞生物学等领域的研究。