Genome-wide identification of short 2′,3′-cyclic phosphate-containing RNAs and their regulation in aging

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2′,3′-cyclic phosphate (cP) at their 3′-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP’s prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.

经核糖核酸酶切割产生的RNA分子，其3'端常携带2',3'-环磷酸（cP）修饰。这类携带cP修饰的RNA（cP-RNAs）构成了转录组中一类隐蔽的组分，由于cP修饰会阻断接头连接反应，标准RNA测序无法捕获这类RNA。本研究针对多种小鼠组织中的短链cP-RNA转录组开展了全基因组分析。借助可特异性靶向测序cP-RNAs的cP-RNA-seq技术，我们鉴定出大量新型cP-RNA物种，这些RNA主要源自细胞质转运RNA（tRNA）、信使RNA（mRNA）及核糖体RNA（rRNA）。对cP-RNA生成过程中底物RNA的加工位点进行解析后发现，在cP-RNA的生物生成过程中，存在高度特异性的胞苷与腺苷位点间的RNA切割事件。cP-RNAs并非均匀分布于底物RNA的全区域，而是源自特定位点，这提示cP-RNAs并非随机降解产物，而是通过受调控的生物生成途径产生的。鉴定出的cP-RNAs在小鼠组织中大量富集，且其表达水平随年龄增长呈下降趋势。本研究对cP-RNA转录组的分析，揭示了一类全新的、丰度颇高的非编码RNA类群，这类RNA的表达可能具有生理功能。