Accurate SHAPE-directed RNA structure determination

Abstract

Almost all RNAs can fold to form extensive base-paired secondary structures. Many of these structures then modulate numerous fundamental elements of gene expression. Deducing these structure-function relationships requires that it be possible to predict RNA secondary structures accurately. However, RNA secondary structure prediction for large RNAs, such that a single predicted structure for a single sequence reliably represents the correct structure, has remained an unsolved problem. Here, we demonstrate that quantitative, nucleotide-resolution information from a SHAPE experiment can be interpreted as a pseudo-free energy change term and used to determine RNA secondary structure with high accuracy. Free energy minimization, by using SHAPE pseudo-free energies, in conjunction with nearest neighbor parameters, predicts the secondary structure of deproteinized Escherichia coli 16S rRNA (>1,300 nt) and a set of smaller RNAs (75-155 nt) with accuracies of up to 96-100%, which are comparable to the best accuracies achievable by comparative sequence analysis.

Fig. 1.

Accuracy of secondary structure prediction for E. coli 16S rRNA by using free energy minimization alone. Base pairs determined by comparative sequence analysis (32) but not predicted by free energy minimization are represented by red x's; predicted pairs not present in the covariation structure are indicated by lines.

Fig. 2.

Analysis of E. coli rRNA structure by SHAPE. (A) Total RNA isolation under nondenaturing conditions and modification with a SHAPE electrophile. (B) Resolution of SHAPE reactivities by capillary electrophoresis. (C) Calculation of normalized SHAPE reactivities by box-plot analysis (31). (D) Histogram of SHAPE data and superposition on the secondary structure for E. coli 23S rRNA.

Fig. 3.

SHAPE data superimposed on domain II of the covariation-based structure (32) of E. coli 23S rRNA. Nucleotides are colored by their SHAPE reactivities; nucleotides with no data are gray; positions at which SHAPE reactivities are not consistent with the covariation structure are enclosed by blue boxes.

Fig. 4.

Accuracy of RNA secondary structures for E. coli 23S rRNA as a function of ΔG_SHAPE pseudo-free energy change parameters.

Fig. 5.

Accuracy of SHAPE-directed secondary structure determination for E. coli 16S rRNA. ΔG_SHAPE parameters were intercept and slope of −0.8 and 2.6 kcal/mol, respectively. Missed base pairs are indicated by red x's; incorrectly predicted base pairs are represented by purple lines. Nucleotides are colored by their SHAPE reactivities. Regions where SHAPE reactivities are not consistent with the accepted phylogenetic structure are indicated with blue boxes. Regions and specific base pairs where the experimental SHAPE information supports local refolding are indicated with green boxes and spheres, respectively.