The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels
The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels
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DOI：
10.1101/610790
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发表时间：
2019-04
影响因子：
14.9
通讯作者：
Matthew R. McFarland;Matthew R. McFarland;Corina D. Keller;Brandon M. Childers;Holly Corrigall;Adélaïde Raguin;Adélaïde Raguin;M. Romano;I. Stansfield
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文献类型：
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作者：
Matthew R. McFarland;Matthew R. McFarland;Corina D. Keller;Brandon M. Childers;Holly Corrigall;Adélaïde Raguin;Adélaïde Raguin;M. Romano;I. Stansfield
在蛋白质合成期间，带电荷的tRNA将氨基酸递送到翻译核糖体，然后通过tRNA合成酶（阿尔斯）再充电。在人类中，突变型阿尔斯引起多种神经系统疾病，但其分子病因学特征不完全。为了理解系统对阿尔斯耗尽的响应，使用多西环素通过tet-off控制来转录调节酵母谷氨阿尔斯基因（GLN 4）。耗尽Gln 4p抑制生长，并诱导GCN 4氨基酸饥饿反应，表明不带电的tRNA积累和GCN 2激酶激活。使用包括阿尔斯再充电的整体翻译模型，模拟Gln 4p耗竭，证实翻译减慢。建模还揭示了Gln 4p耗尽引起负反馈，其将Gln-tRNAGln的翻译需求与阿尔斯再充电能力相匹配。这维持了正常的带电荷的tRNAGln水平，尽管Gln 4 p耗尽，使用tRNA北方印迹实验证实。模型分析解决了Gln 4p耗尽触发GCN 4应答的悖论，尽管维持tRNAGln充电水平，揭示了阿尔斯群体可以在氨酰化期间螯合游离的、不带电荷的tRNA。Gln 4p缺失降低了这种螯合能力，允许不带电荷的tRNAGln与Gcn 2激酶相互作用。这项研究为突变阿尔斯疾病的病因提供了新的线索，并解释了阿尔斯对不带电荷的tRNA的隔离如何在非饥饿条件下阻止GCN 4的激活。
During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.