Tsukamoto Yusuke, Nakamura Yumi, Hirata Makoto, Sakate Ryuichi, Kimura Tomonori
National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN).
National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)
RNA. 2022 Oct 25;29(1):111-22. doi: 10.1261/rna.079323.122.
Each transfer RNA (tRNA) is aminoacylated (charged) with a genetic codon-specific amino acid at its 3' end. Charged tRNAs are primarily used for translation, whereas fluctuations in charged tRNA fractions are known to reflect cellular response to stress. Here we report the development of individual tRNA-acylation using PCR (i-tRAP), a convenient PCR-based method that can specifically quantify individual tRNA charging ratio. In this i-tRAP method, demethylases remove base methylations which are problematic for reverse transcription reaction, and β-elimination reaction specifically removes the 3' end of adenine residue in uncharged tRNA. Subsequent TaqMan MGB qRT-PCR can distinguish between cDNA of charged tRNA and uncharged tRNA. By using this method, we revealed that the charging ratio of tRNAGln(CUG) was changed in response to amino acid starvation and also the charging ratio of tRNAGln(CUG) in senescent cells was lower than in young cells under starvation conditions. i-tRAP can be applicable to the quantification of charging ratio of various tRNAs, and provides a simple and convenient method for analyzing tRNA charging.
每个转运RNA（tRNA）在其3'末端被特定遗传密码子的氨基酸进行氨酰化（充电）。氨酰化的tRNA主要用于翻译，而氨酰化tRNA比例的波动已知可反映细胞对压力的反应。在这里，我们报告了使用PCR进行单个tRNA氨酰化分析（i-tRAP）的方法，这是一种基于PCR的便捷方法，可特异性定量单个tRNA的氨酰化比率。在这种i-tRAP方法中，去甲基化酶去除对逆转录反应有问题的碱基甲基化，β-消除反应特异性去除未氨酰化tRNA中腺嘌呤残基的3'末端。随后的TaqMan MGB qRT-PCR可以区分氨酰化tRNA和未氨酰化tRNA的cDNA。通过使用这种方法，我们发现tRNAGln（CUG）的氨酰化比率会因氨基酸饥饿而改变，并且在饥饿条件下，衰老细胞中tRNAGln（CUG）的氨酰化比率低于年轻细胞。i-tRAP可用于定量各种tRNA的氨酰化比率，并为分析tRNA氨酰化提供了一种简单便捷的方法。
