ABSTRACT

Under adverse conditions, tRNAs are processed into fragments called tRNA-derived stress-induced RNAs (tiRNAs) by stress-responsive ribonucleases (RNases) such as angiogenin (ANG). Recent studies have reported several biological functions of synthetic tiRNAs lacking post-transcriptional modifications found on endogenous tiRNAs. Here we describe a simple and reproducible method to efficiently isolate ANG-cleaved tiRNAs from endogenous tRNAs. Using this in vitro method, more than 50% of mature tRNAs are cleaved into tiRNAs which can be enriched using complementary oligonucleotides. Using this method, the yield of isolated endogenous 5ʹ-tiRNA^Gly-GCC was increased about fivefold compared to when tiRNAs were obtained by cellular treatment of ANG. Although the non-specific ribonuclease activity of ANG is much lower than that of RNase A, we show that ANG cleaves physiologically folded tRNAs as efficiently as bovine RNase A. These results suggest that ANG is highly specialized to cleave physiologically folded tRNAs. Our method will greatly facilitate the analysis of endogenous tiRNAs to elucidate the physiological functions of ANG.

摘要

在不利条件下，tRNA 被血管生成素 (ANG) 等应激反应性核糖核酸酶 (RNase) 加工成称为 tRNA 衍生的应激诱导 RNA (tiRNA) 的片段。最近的研究报告了合成 tiRNA 的几种生物学功能，这些 tiRNA 缺乏在内源性 tiRNA 上发现的转录后修饰。在这里，我们描述了一种简单且可重复的方法，可以有效地将 ANG 切割的 tiRNA 与内源性 tRNA 分离。使用这种体外方法，超过 50% 的成熟 tRNA 被切割成 tiRNA，可以使用互补寡核苷酸进行富集。使用这种方法，分离的内源性 5'-tiRNA ^{Gly-GCC的产量}与通过 ANG 的细胞处理获得 tiRNA 时相比，增加了约五倍。尽管 ANG 的非特异性核糖核酸酶活性远低于 RNase A，但我们表明 ANG 与牛 RNase A 一样有效地切割生理折叠的 tRNA。这些结果表明 ANG 高度专门用于切割生理折叠的 tRNA。我们的方法将极大地促进内源性 tiRNA 的分析，以阐明 ANG 的生理功能。