Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.
Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.
复制标题
同时表征细胞RNA结构和功能与细胞内形状隔离。
DOI：
10.1093/nar/gkv879
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发表时间：
2016-01-29
影响因子：
14.9
通讯作者：
Lucks JB
中科院分区：
文献类型：
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作者：
Watters KE;Abbott TR;Lucks JB
许多非编码RNA形成与细胞机器相互作用以控制基因表达的结构。分子和合成生物学的一个中心目标是揭示连接RNA结构和功能的设计原则，以理解和设计这种关系。在这里，我们报告了一种称为细胞内SHAPE-Seq的简单、高通量方法，该方法将RNA结构的细胞内探测与基因表达的测量相结合，以同时表征细菌细胞中的RNA结构和功能。我们使用细胞内SHAPE-Seq来研究大肠杆菌中调节翻译的两种RNA机制的结构-功能关系。我们发现，参与RNA-RNA相互作用的核苷酸是高度可访问的，当它们的结合伴侣是缺席的，RNA结构的变化，由于RNA-RNA相互作用可以定量相关的基因表达的变化。我们还表征了三种内源性表达的非编码RNA的细胞结构：5S rRNA，RNase P和btuB核糖开关。最后，细胞内和体外折叠RNA结构之间的比较显示，合成RNA具有显着的相似性，但参与复杂细胞相互作用的RNA存在显着差异。因此，细胞内SHAPE-Seq代表了生物学家和工程师揭示细胞中RNA序列，结构和功能之间关系的一种容易接近的工具。
Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure–function relationship of two RNA mechanisms that regulate translation in Escherichia coli. We find that nucleotides that participate in RNA–RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA–RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell.