A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Abstract

Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

Figure 1.

Comparison of SHAPE reagents under ex vivo conditions. (A) Boxplot of SHAPE−MaP mutation frequencies for E. coli 16S and 23S rRNAs probed ex vivo after deproteinization. Box plots span the interquartile range (from Q1 to Q3). The grey area spans from the median in the DMSO sample (control) to the median in the NAI sample (reference). (B) ROC curves for all tested SHAPE reagents, calculated with respect to the accepted phylogenetically-inferred 16S and 23S rRNA structures from the Comparative RNA Web (30). The inset reports the area under the curve (AUC) for each compound. (C) Sample of SHAPE−MaP mutation frequencies for all tested compounds across a region spanning nucleotides 567 to 884 of E. coli 16S rRNA. Colored bases are those whose mutation frequencies exceed by 2-fold the median mutation frequency in the analyzed region. The accepted structure is reported as an arc plot.

Figure 2.

Comparison of SHAPE reagents under in vivo conditions. (A) Boxplot of SHAPE−MaP mutation frequencies for E. coli 16S and 23S rRNAs probed in vivo. Box plots span the interquartile range (from Q1 to Q3). The grey area spans from the median in the DMSO sample (control) to the median in the NAI sample (reference). (B) ROC curves for all tested SHAPE reagents, calculated on solvent-exposed residues in the crystal structure of the E. coli ribosome (PDB: 5IT8), with respect to the accepted phylogenetically-inferred 16S and 23S rRNA structures from the Comparative RNA Web (30). The inset reports the AUC for each compound. (C) Median in vivo SHAPE-MaP mutation frequencies across all stem–loops in the accepted E. coli 16S and 23S rRNA structures. Bases are numbered relatively to the loop. Positions –3 to –1 correspond to stem bases, while positions 0 and +1 correspond to loop bases. (D) Ratio between the median loop mutation frequency and the median stem mutation frequency, calculated on all stem–loops from C.

Figure 3.

2A3 successfully queries regions of the ribosome that are blind to other reagents. (A) Sample of SHAPE−MaP mutation frequencies for all tested compounds across a region spanning nucleotides 990 to 1162 of E. coli 23S rRNA. Colored bases are those whose mutation frequencies exceed by 2-fold the median mutation frequency in the analyzed region. The accepted structure is reported as an arc plot. The hyperreactive residue U₁₀₃₂ and the unreactive stretch of 19 nucleotides are respectively marked in red and blue. (B) Three-dimensional model of the domain depicted in A, colored by B factor (PDB: 5IT8). The inset zooms on the helical region containing the 2A3-hyperreactive residue U₁₀₃₂.

Figure 4.

2A3 outperforms NAI at experimentally-driven RNA structure modeling. (A) Grid search (jackknifing) of optimal slope/intercept value pairs for E. coli 16S and 23S rRNAs in vivo probing data for NAI and 2A3. Values represent the geometric mean of sensitivity and PPV for the secondary structures predicted using each slope/intercept value pair. The chosen value pair is boxed in green. (B) Arc plot comparison of E. coli 16S rRNA reference structure (top), and structure inferred using either NAI-derived or 2A3-derived restraints (bottom). Black/green arcs correspond to correctly predicted base-pairs, violet arcs to non-predicted base-pairs and red arcs to mispredicted base-pairs. PPV and sensitivity for each prediction are indicated.

Figure 5.

Mechanism of 2A3 reaction with RNA. Reaction of 2A3 with the 2′-OH of structurally-flexible RNA residues, resulting in the formation of a 2′-O-adduct.