Translation in amino acid-poor environments is limited by tRNAGln charging. Translation in amino acid-poor environments is limited by tRNAGln charging

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as the key sensors of glutamine availability in mammalian cells. Overall design: Mouse embryonic fibroblasts were cultured for 6h with or without amino acids in the presence of glutaminase inhibitor (CB-839) or vehicle (DMSO). RNA was extracted from cells and treated with either 10mM sodium periodite ("oxidized") or sodium chloride ("non-oxidized) prior to sequencing library preparation to calculate tRNA charging ratios.

氨基酸供给不足会导致空载转运RNA（uncharged tRNAs）积累，后者可结合并激活GCN2激酶，进而抑制细胞整体翻译过程。本研究发现，当细胞外氨基酸可利用性受损时，谷氨酰胺特异性转运RNA（tRNAGln）会选择性地处于空载状态；与之相反，其余所有转运RNA仍能维持其对应氨基酸的氨酰化修饰，且该过程依赖于完整的溶酶体功能。除激活GCN2激酶并降低整体翻译水平外，氨基酸剥夺处理的细胞中tRNAGln的氨酰化水平降低，还会特异性耗竭带有聚谷氨酰胺（polyglutamine）重复序列的蛋白质，包括核心结合因子α1、中介体亚基12、转录辅激活因子CBP以及TATA盒结合蛋白。向氨基酸剥夺的细胞添加外源性谷氨酰胺或谷氨酰胺酶抑制剂，可恢复tRNAGln的氨酰化水平以及聚谷氨酰胺重复序列蛋白的丰度。综上，本研究结果表明，GCN2激酶的激活与聚谷氨酰胺编码转录本的翻译，是哺乳动物细胞中谷氨酰胺可利用性的关键感受器。实验设计概述：将小鼠胚胎成纤维细胞置于添加谷氨酰胺酶抑制剂CB-839或载体二甲基亚砜（DMSO）的培养基中，分别在含氨基酸与无氨基酸的条件下培养6小时。随后从细胞中提取RNA，并在构建测序文库前分别用10mM高碘酸钠（"氧化组"）或氯化钠（"非氧化组"）处理，以计算tRNA的氨酰化比率。