The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA
The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA
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DOI：
10.4161/cc.10.15.16347
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发表时间：
2011-08-01
期刊：
影响因子：
4.3
通讯作者：
O'Reilly, Michael A.
中科院分区：
文献类型：
--
作者：
Gewandter, Jennifer S.;Bambara, Robert A.;O'Reilly, Michael A.
DNA 损伤、复制叉停滞、mRNA 剪接错误和营养物质的可用性会激活特定的磷脂酰肌醇 3 激酶样激酶 (PIKK)，进而磷酸化下游靶标，例如丝氨酸 15 上的 p53。PIKK 蛋白 ATM 和 ATR 对特定 DNA 损伤做出反应，而 SMG1 对 mRNA 剪接错误以及细胞受到损伤时做出反应。 暴露于遗传毒性应激。然而，基因毒性应激是否通过特定类型的 DNA 损伤或 RNA 损伤激活 SMG1 仍知之甚少。在这里，我们证明，针对 mRNA 监视蛋白 SMG1、Upf1、Upf2 或 PIKK 蛋白 ATM 的 siRNA 寡核苷酸可减弱高氧（高氧）损伤细胞中的 p53 (ser15) 磷酸化，高氧是一种损害核苷酸的持续氧化应激模型。相比之下，SMG1 或 ATM 的缺失（而非 Upf1 或 Upf2）会降低 p53 (ser15) 磷酸化，以响应内切核酸酶 I-PpoI 表达产生的 DNA 双链断裂。为了确定 p53 的 SMG1 依赖性激活是否是对氧化 mRNA 损伤的反应，体外转录的编码绿色荧光蛋白 (GFP) 的 mRNA 通过芬顿化学氧化并转染到细胞中。虽然 GFP mRNA 的氧化导致 mRNA 的剂量依赖性断裂并减少 GFP 的表达，但它不会刺激 p53 或 p53 靶基因 p21。这些发现证实，SMG1 响应 DNA 双链断裂而激活 p53，独立于 RNA 监视蛋白 Upf1 或 Upf2；然而，这些蛋白质可以刺激 p53 响应氧化应激，但不一定会氧化 RNA。
DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.