Detection of RNA–Protein Interactions in Living Cells with SHAPE

Abstract

SHAPE-MaP is unique among RNA structure probing strategies in that it both measures flexibility at single-nucleotide resolution and quantifies the uncertainties in these measurements. We report a straightforward analytical framework that incorporates these uncertainties to enable detection of RNA structural differences between any two states, and we use it here to detect RNA-protein interactions in healthy mouse trophoblast stem cells. We validate this approach by analysis of three model cytoplasmic and nuclear ribonucleoprotein complexes, in 2-minute in-cell probing experiments. In contrast, data produced by alternative in-cell SHAPE probing methods correlate poorly (r = 0.2) with those generated by SHAPE-MaP and do not yield accurate signals for RNA-protein interactions. We then examine RNA-protein and RNA-substrate interactions in the RNase MRP complex and, by comparing in-cell interaction sites with disease-associated mutations, characterize these non-coding mutations in terms of molecular phenotype. Together, these results reveal that SHAPE-MaP can define true interaction sites and infer RNA functions under native cellular conditions with limited pre-existing knowledge of the proteins or RNAs involved.

Comparison of SHAPE reagents for mapping RNA structures inside living cells

“…However, recent reports have suggested that 1M7, an isatoic anhydride reagent related to NMIA, was able to robustly modify both ribosomal RNA as well as mRNAs inside both mammalian and bacterial cells (McGinnis et al 2015;Smola et al 2015;Takahashi et al 2016;Watters et al 2016). These apparently contradictory results stimulated us to further investigate the differences between the two types of SHAPE reagents.…”

“…Published work has reported in vivo modification of RNA in E. coli cells; however, no cDNA truncation blots were shown (McGinnis et al 2015;Smola et al 2015;Takahashi et al 2016). To address the possibility that our lack of in vivo signal using 1M7 is due to the type of cells used, we repeated the SHAPE experiment in HST08 E. coli cells following precisely the published protocols for cell culture, RNA modification, and RNA extraction (McGinnis et al 2015).…”

“…RNA was modified and collected from mESCs using the same conditions as previously published for mammalian cell modification (Smola et al 2015). Old media was removed and cells were washed once with PBS.…”

“…Fourth, published data for each experimental type were produced in different cell types from differentially purified RNA inputs. Despite these substantial differences, a recent report compared these two methods (Smola et al 2015) without attempting to control for the aforementioned differences in SHAPE reagents and method, and without direct experimental testing of the two methods.…”

Genome-Wide Analysis of RNA Secondary Structure

“…Mutational profiling analysis is particularly useful for identifying correlations between two positions within a single RNA but has the drawback that mutational profiling analysis requires much deeper sequencing to confidently call a nucleotide single stranded because there may be mutations incorporated during library preparation unrelated to a SHAPE adduct. This method has the potential to become an in vivo probing method by incorporating one of the cell-permeable SHAPE reagents (102,105).…”