In vivo cleavage map illuminates the central role of RNase E in coding and noncoding RNA pathways

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs, whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3'-fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post- transcriptional regulators. Overall design: 8 total RNA samples from Salmonella WT and rne-3071(rne-TS) strains grown at 28°C with and without heat treatment (44°C) for 30min, in duplicate.

解析RNA的加工与周转过程，有赖于对活细胞内主要核糖核酸内切酶（endoribonuclease）切割行为的认知。本研究借助TIER-Seq（瞬时灭活核糖核酸内切酶后衔接RNA测序），对肠炎沙门氏菌（Salmonella enterica）中必需核糖核酸内切酶RNase E的切割产物进行了全景分析。最显著的切割特征为：单链区段下游第2个核苷酸位点存在尿苷（uridine），我们通过结构分析将其阐释为可促进RNase E催化反应的关键识别元件。本研究结果揭示了细菌调控型小RNA的一条重要生物发生途径：RNase E与RNA分子伴侣（RNA chaperone）Hfq协同作用，从各类前体RNA中释放出稳定的3'端片段。在体外重现该过程时，Hfq可引导RNase E切割可与mRNA靶标结合的代表性小RNA前体。在活体状态下，该加工过程对于靶标调控不可或缺。本研究的发现揭示了一类主要转录后调控因子的通用成熟机制。实验整体设计：共采集8份总RNA样本，分别来自28℃下培养、经44℃热激处理30分钟或未做热激处理的肠炎沙门氏菌野生型（WT）与rne-3071（rne-TS）菌株，每个组别均设置两份生物学重复。