McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX, 78712, USA.
Department of Operations Research and Financial Engineering, Princeton University, Sherrerd Hall, Charlton St., Princeton, NJ, 08544, USA.
Nat Commun. 2018 Oct 4;9(1):4084. doi: 10.1038/s41467-018-06207-z.
Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.
在此，我们介绍了一种高通量方法 INTERFACE，用于揭示连续 RNA 核苷酸在体内建立分子间 RNA 相互作用的能力，旨在对细胞内 RNA 的功能进行特征分析。INTERFACE 通过将区域杂交检测与可通过 RNA-seq 测量的转录延伸产物相结合，实现了对无限数量区域的同时可及性检测。我们在存在和不存在全局调节剂 Hfq 的情况下，对 71 个经过验证但在很大程度上机制尚未阐明的大肠杆菌 sRNA 中的 900 多个 RNA 界面进行了分析，发现三分之二的测试 sRNA 具有依赖 Hfq 的区域。此外，我们还确定了体内杂交模式，这些模式标志着功能区域，以揭示 mRNA 靶标。通过这种方式，我们对 25 个 mRNA 靶标进行了生化验证，其中许多靶标未被通常测试的排名最高的计算预测所捕获。我们还在 GlmY 终止子内发现了直接的 mRNA 结合活性，突出了高通量 RNA 可及性数据的信息价值。
