Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq

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 struc-ture to function to understand and engineer this re-lationship. 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 rela-tionship of two RNA mechanisms that regulate trans-lation 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 inter-actions can be quantitatively correlated to changes in gene expression. We also characterize the cellu-lar structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB ri-boswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differ-ences 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, struc-ture and function of RNAs in the cell