Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK.
Nucleic Acids Res. 2013 Apr;41(7):4185-97. doi: 10.1093/nar/gkt129. Epub 2013 Mar 5.
Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD(+)-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.
活性 SIRT1 调节因子（AROS）结合并上调 SIRT1，这是一种 NAD（+）依赖性去乙酰化酶。此外，AROS 还结合 RPS19，这是一种结构核糖体蛋白，它也参与核糖体生物发生，与多种疾病状态有关。AROS 与核糖体生物发生和功能的关系尚不清楚。使用人类细胞，我们现在表明 AROS 定位于（i）核仁（ii）细胞质核糖体。通过共聚焦免疫荧光术对内源性蛋白进行的免疫荧光验证以及通过 RNAi 进行的 AROS 耗竭证实了核仁蛋白的共定位。通过蔗糖密度分级分离和免疫沉淀分析了 AROS 与细胞质核糖体的关联，结果表明 AROS 选择性地与 40S 核糖体亚基以及多核糖体结合。通过 RNAi 介导的 AROS 耗竭导致核糖体生物发生缺陷，前体核糖体 RNA 加工异常，40S 亚基核糖体 RNA 和 40S 核糖体蛋白（包括 RPS19）减少。总之，这导致 40S 亚基和翻译多核糖体减少，与整体细胞蛋白质合成减少相关。有趣的是，AROS 的敲低也导致 eIF2α 磷酸化的功能显著增加。总的来说，我们的结果表明 AROS 是参与核糖体生物发生和核糖体功能的一个因素。
