Inhibition of nonsense-mediated RNA decay by ER stress.
Inhibition of nonsense-mediated RNA decay by ER stress.
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DOI：
10.1261/rna.058040.116
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发表时间：
2017-03
期刊：
影响因子：
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通讯作者：
Zheng S
中科院分区：
文献类型：
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作者：
Li Z;Vuong JK;Zhang M;Stork C;Zheng S
无义介导的RNA衰变（NMD）选择性降解含有提前终止密码子（PTC）的突变和异常加工的转录物。细胞NMD活性通常使用含有外源性PTC的报告基因来评估。我们克服了测定内源性靶标的一些固有问题，并开发了一种广泛适用的策略来可靠且容易地监测细胞NMD活性的变化。我们的新方法在遗传学上被验证用于区分NMD调控与转录控制和可变剪接调控，并且出乎意料地公开了NMD靶点对NMD抑制的不同敏感性。应用这种强大的筛选方法，我们确定了NMD抑制应激源，但也发现NMD失活并不普遍的细胞压力。我们的方法的高灵敏度和宽的动态范围揭示了NMD抑制，内质网（ER）的压力，和多核糖体解体后毒胡萝卜素治疗的时间和剂量依赖性的方式之间的强相关性。我们发现几乎没有证据表明钙信号介导毒胡萝卜素诱导的NMD抑制。相反，我们发现在毒胡萝卜素激活的三种未折叠蛋白反应（UPR）途径中，NMD抑制主要需要蛋白激酶RNA样内质网激酶（PERK）。最后，我们发现，ER应激复合TDP-43耗竭的上调NMD亚型，已牵连在肌萎缩侧索硬化症和额颞叶痴呆的发病机制，和ER应激的累加效应被完全阻断PERK缺陷。
Nonsense-mediated RNA decay (NMD) selectively degrades mutated and aberrantly processed transcripts that contain premature termination codons (PTC). Cellular NMD activity is typically assessed using exogenous PTC-containing reporters. We overcame some inherently problematic aspects of assaying endogenous targets and developed a broadly applicable strategy to reliably and easily monitor changes in cellular NMD activity. Our new method was genetically validated for distinguishing NMD regulation from transcriptional control and alternative splicing regulation, and unexpectedly disclosed a different sensitivity of NMD targets to NMD inhibition. Applying this robust method for screening, we identified NMD-inhibiting stressors but also found that NMD inactivation was not universal to cellular stresses. The high sensitivity and broad dynamic range of our method revealed a strong correlation between NMD inhibition, endoplasmic reticulum (ER) stress, and polysome disassembly upon thapsigargin treatment in a temporal and dose-dependent manner. We found little evidence of calcium signaling mediating thapsigargin-induced NMD inhibition. Instead, we discovered that of the three unfolded protein response (UPR) pathways activated by thapsigargin, mainly protein kinase RNA-like endoplasmic reticulum kinase (PERK) was required for NMD inhibition. Finally, we showed that ER stress compounded TDP-43 depletion in the up-regulation of NMD isoforms that had been implicated in the pathogenic mechanisms of amyotrophic lateral sclerosis and frontotemporal dementia, and that the additive effect of ER stress was completely blocked by PERK deficiency.