In-cell RNA structure probing with SHAPE-MaP

Abstract

This protocol is an extension to: Nat. Protoc. 10, 1643-1669 (2015); doi:10.1038/nprot.2015.103; published online 01 October 2015RNAs 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.

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo

“…In the previous DMS Structure-seq data, the cleavage site leads to reverse transcription stalling, and causes a skewed DMS reactivity profile due to false positive signals (Figure 2A). In our CAP-STRUCTURE-seq data, these degradation signals were excluded ( Figure 2B), thereby overcoming the limitations of previous methods that include degradation products (Supplementary Figure S1) (8,10,13,41,42). Overall, these data demonstrate that CAP-STRUCTURE-seq can accurately identify in vivo structures of intact mRNAs.…”

Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

“…In contrast, MaPseq has the advantage that multiple reactions on one mRNA molecule can be detected. MaPseq is used in combination with DMS [28] and SHAPE [27,29].Recently, it was discovered that under alkaline conditions-in contrast to neutral pH-DMS probes all four nucleotides [30]. Mustoe, et al [30] used an alkaline buffer for DMS probing of E. coli and mammalian cells which had a cytosol with neutral pH.…”

“…In contrast, MaPseq has the advantage that multiple reactions on one mRNA molecule can be detected. MaPseq is used in combination with DMS [28] and SHAPE [27,29].…”

Targeting the Oncogenic Long Non-coding RNA SLNCR1 by Blocking Its Sequence-Specific Binding to the Androgen Receptor

“…MSTCs have undergone limited passages outside of the patient and therefore closely reflect the genetics and relative gene expression patterns of the tumor. We probed the structure of SLNCR1 using two cell-permeable reagents: the SHAPE reagent 5-nitroisatoic anhydride (5NIA), which reacts broadly with all four nucleotides, and the methylating agent dimethyl sulfate (DMS), which reacts most strongly with adenosine and cytidine (Busan et al, 2019;Smola and Weeks, 2018). We probed the nucleotide flexibilities of SLNCR1 both within WM1976 cells (in cell) and after gentle extraction of the RNA from WM1976 cells into a protein-free environment (cell free).…”