Laboratory for Protein Conformation Diseases, RIKEN Brain Science Institute, Hirosawa 2-1, Wako, Saitama 351-0198, Japan.
Laboratory for Protein Conformation Diseases, RIKEN Brain Science Institute, Hirosawa 2-1, Wako, Saitama 351-0198, Japan.
Cell Rep. 2018 Apr 10;23(2):608-621. doi: 10.1016/j.celrep.2018.03.035.
In the ribosome complex, tRNA is a critical element of mRNA translation. A rich repertoire of cell regulation is hypothesized to occur during the recruitment of specific tRNAs in polypeptide formation. However, this basic question in nascent chain biology remains unaddressed due to the lack of technologies to report the complete tRNA complement inside ribosomes during active translation. Here, we characterize a technique for profiling ribosome-embedded tRNA and their modifications. With this method, we generated a comprehensive survey of the quantity and quality of intra-ribosomal tRNAs. In cells under environmental stress, we show that methionine tRNA inside ribosomes is a robust biomarker for the impairment of translation initiation or elongation steps. Concurrent tRNA/mRNA ribosome profiling revealed a stress-dependent incorporation of damaged and uncharged tRNAs into ribosomes causing translation arrest. Thus, tRNA ribosome profiling can provide insights on translation control mechanisms in diverse biological contexts.
在核糖体复合物中，tRNA 是 mRNA 翻译的关键元件。人们假设，在多肽形成过程中，特定 tRNA 的招募会发生丰富的细胞调控。然而，由于缺乏在活跃翻译过程中报告核糖体中完整 tRNA 成分的技术，这个新生链生物学中的基本问题仍然没有得到解决。在这里，我们描述了一种用于分析核糖体中嵌入的 tRNA 及其修饰的技术。通过这种方法，我们全面调查了核糖体中内源性 tRNA 的数量和质量。在受到环境压力的细胞中，我们表明核糖体中的蛋氨酸 tRNA 是翻译起始或延伸步骤受损的稳健生物标志物。同时进行的 tRNA/mRNA 核糖体分析显示，应激依赖性地将受损和未加载的 tRNA 掺入核糖体中，导致翻译停滞。因此，tRNA 核糖体分析可以为不同生物背景下的翻译控制机制提供深入了解。
