Department of Biomedical Sciences, University at Albany, State University of New York, Rensselaer, New York 12144, USA.
Mol Cancer Res. 2011 Apr;9(4):462-75. doi: 10.1158/1541-7786.MCR-10-0473. Epub 2011 Feb 22.
Protein synthesis and degradation are posttranscriptional pathways used by cells to regulate protein levels. We have developed a systems biology approach to identify targets of posttranscriptional regulation and we have employed this system in Saccharomyces cerevisiae to study the DNA damage response. We present evidence that 50% to 75% of the transcripts induced by alkylation damage are regulated posttranscriptionally. Significantly, we demonstrate that two transcriptionally-induced DNA damage response genes, RNR1 and RNR4, fail to show soluble protein level increases after DNA damage. To determine one of the associated mechanisms of posttranscriptional regulation, we tracked ribonucleotide reductase 1 (Rnr1) protein levels during the DNA damage response. We show that RNR1 is actively translated after damage and that a large fraction of the corresponding Rnr1 protein is packaged into a membrane-bound structure and transported to the vacuole for degradation, with these last two steps dependent on autophagy proteins. We found that inhibition of target of rapamycin (TOR) signaling and subsequent induction of autophagy promoted an increase in targeting of Rnr1 to the vacuole and a decrease in soluble Rnr1 protein levels. In addition, we demonstrate that defects in autophagy result in an increase in soluble Rnr1 protein levels and a DNA damage phenotype. Our results highlight roles for autophagy and TOR signaling in regulating a specific protein and demonstrate the importance of these pathways in optimizing the DNA damage response.
蛋白质合成和降解是细胞用来调节蛋白质水平的转录后途径。我们开发了一种系统生物学方法来鉴定转录后调控的靶标，并将该系统应用于酿酒酵母中研究 DNA 损伤反应。我们提供的证据表明，烷基化损伤诱导的转录本中有 50%至 75%受到转录后调控。重要的是，我们证明了两个转录诱导的 DNA 损伤反应基因 RNR1 和 RNR4 在 DNA 损伤后未能显示可溶性蛋白水平的增加。为了确定转录后调控的一种相关机制，我们在 DNA 损伤反应过程中跟踪了核核苷酸还原酶 1（Rnr1）蛋白水平。我们表明 RNR1 在损伤后被积极翻译，并且相应的 Rnr1 蛋白的很大一部分被包装到膜结合结构中，并被运送到液泡中进行降解，后两个步骤依赖于自噬蛋白。我们发现，抑制雷帕霉素（TOR）信号转导并随后诱导自噬会促进 Rnr1 靶向液泡的增加和可溶性 Rnr1 蛋白水平的降低。此外，我们证明自噬缺陷会导致可溶性 Rnr1 蛋白水平升高和 DNA 损伤表型。我们的结果强调了自噬和 TOR 信号在调节特定蛋白质中的作用，并证明了这些途径在优化 DNA 损伤反应中的重要性。
