Eukaryotic cells equip robust systems to respond to stress conditions. In stressed mammalian cells, angiogenin endoribonuclease cleaves anticodon-loops of tRNAs to generate tRNA halves termed tRNA-derived stress-induced RNAs (tiRNAs), which promote stress granule formation and regulate translation. The 5′-tiRNAs (5′-tRNA halves) contain a 2′,3′-cyclic phosphate (cP) and thus belong to cP-containing RNAs (cP-RNAs). The cP-RNAs form a hidden layer of the transcriptome because standard RNA-seq cannot amplify and sequence them. In this study, we performed genome-wide analyses of short cP-RNA transcriptome in oxidative stress-exposed human cells. Using cP-RNA-seq that can specifically sequence cP-RNAs, we identified tiRNAs and numerous other cP-RNAs that are mainly derived from rRNAs and mRNAs. Although tiRNAs were produced from a wide variety of tRNA species, abundant species of tiRNAs were derived from a focal-specific subset of tRNAs. Regarding rRNA- and mRNA-derived cP-RNAs, determination of the processing sites of substrate RNAs revealed highly specific RNA cleavage events between pyrimidines and adenosine in generation of those cP-RNAs. Those cP-RNAs were derived from specific loci of substrate RNAs rather than from the overall region, implying that cP-RNAs are produced by regulated biogenesis pathways and not by random degradation events. We experimentally confirmed the identified sequences to be expressed as cP-RNAs in the cells, and their expressions were upregulated upon induction of oxidative stress. These analyses of the cP-RNA transcriptome unravel an abundant class of short ncRNAs that accumulate in cells under oxidative stress.

真核细胞配备了强大的系统来应对压力条件。在受压的哺乳动物细胞中，血管生成素内切核糖核酸酶切割tRNA的反密码子环，以生成称为tRNA的应力诱导RNA（tiRNA）的tRNA一半，从而促进应力颗粒的形成并调节翻译。5'-tiRNA（5'-tRNA一半）包含2'，3'-环状磷酸酯（cP），因此属于含cP的RNA（cP-RNA）。cP-RNA形成了转录组的隐藏层，因为标准RNA-seq无法对其进行扩增和测序。在这项研究中，我们对暴露于氧化应激的人类细胞中的短cP-RNA转录组进行了全基因组分析。使用可以对cP-RNA进行特异性测序的cP-RNA-seq，我们鉴定了tiRNA和许多其他主要来源于rRNA和mRNA的cP-RNA。尽管tiRNA是由多种tRNA产生的，但丰富的tiRNA种类却是从tRNA的特定部位提取的。关于rRNA和mRNA衍生的cP-RNA，底物RNA加工位点的确定揭示了在这些cP-RNA的产生中嘧啶和腺苷之间的高特异性RNA裂解事件。这些cP-RNA来源于底物RNA的特定位点，而不是整个区域，这意味着cP-RNA是通过调控的生物发生途径而不是随机降解事件产生的。我们实验证实了所鉴定的序列在细胞中以cP-RNA的形式表达，并且它们的表达在诱导氧化应激后被上调。