This protocol is an extension to: Nat. Protoc. 10, 1643–1669 (2015); doi:10.1038/nprot.2015.103; published online 01 October 2015
RNAs play key roles in many cellular processes. The underlying structure of RNA is an important determinant of how transcripts function, are processed, and interact with RNA-binding proteins and ligands. RNA structure analysis by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) takes advantage of the reactivity of small electrophilic chemical probes that react with the 2′-hydroxyl group to assess RNA structure at nucleotide resolution. When coupled with mutational profiling (MaP), in which modified nucleotides are detected as internal miscodings during reverse transcription and then read out by massively parallel sequencing, SHAPE yields quantitative per-nucleotide measurements of RNA structure. Here, we provide an extension to our previous in vitro SHAPE-MaP protocol with detailed guidance for undertaking and analyzing SHAPE-MaP probing experiments in live cells. The MaP strategy works for both abundant-transcriptome experiments and for cellular RNAs of low to moderate abundance, which are not well examined by whole-transcriptome methods. In-cell SHAPE-MaP, performed in roughly 3 d, can be applied in cell types ranging from bacteria to cultured mammalian cells and is compatible with a variety of structure-probing reagents. We detail several strategies by which in-cell SHAPE-MaP can inform new biological hypotheses and emphasize downstream analyses that reveal sequence or structure motifs important for RNA interactions in cells.
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Work in our lab focused on developing quantitative and biophysically rigorous RNA structure-probing technologies is supported by the National Institutes of Health (NIH; R35 GM122532 and R01 AI068462). M.J.S. was a Graduate Research Fellow of the National Science Foundation (DGE-1144081) and was supported in part by an NIH training grant in molecular and cellular biophysics (T32 GM08570). We are indebted to the Calabrese laboratory at the University of North Carolina at Chapel Hill for assistance in developing in-cell probing strategies and to members of the Weeks laboratory for thoughtful feedback regarding the analysis algorithms and strategies described here.
K.M.W. has equity ownership in and serves as an advisor to Ribometrix, to which SHAPE-MaP technologies have been licensed. M.J.S. is an employee of Ribometrix.
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Smola, M., Weeks, K. In-cell RNA structure probing with SHAPE-MaP. Nat Protoc 13, 1181–1195 (2018). https://doi.org/10.1038/nprot.2018.010
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