Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP

Abstract

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that "jumps" across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

Figure 1:. SHAPE-JuMP overview.

RNA is treated with a bivalent SHAPE crosslinker (red), which covalently links proximal nucleotides. Reverse transcription using an engineered jumping, or crosslink-traversing, polymerase records the crosslinked site as a deletion in the cDNA (blue). The cDNA is sequenced and aligned to the reference RNA sequence to identify deletion sites and, thereby, crosslinked nucleotides.

Figure 2:. TBIA mechanism and characterization.

(A) Reaction of TBIA with RNA yields both crosslinks and mono-adducts. IA strictly forms mono-adducts. (B) Reaction of the RNase P RNA with no reagent (–), IA (mono-adduct), or TBIA (crosslinking reagent), visualized by denaturing electrophoresis. (C) Hydrolysis of TBIA in buffer. Reaction was monitored by UV absorbance at 296 nm; absorbance as a function of time was fit to a double exponential rate equation.

Figure 3:. Identification of reverse transcriptase enzymes with the ability to jump RNA crosslinks.

Comparison of crosslink detection in the RNase P RNA by different RT enzymes. Deletion rates for RNAs treated with TBIA and mono adduct-forming NMIA reagents are red and blue, respectively.

Figure 4:. SHAPE-JuMP deletion detection as a function of intervening sequence length and through-space distance.

(A) Deletion rates of a given length due to treatment with monoadduct-forming IA and crosslinker TBIA for the RNase P RNA. Deletion rates are normalized to sum to 1. (B) Distances between nucleotides that mediate TBIA-induced deletions. The most frequent three percent of deletion rates are shown with colored lines. Random internucleotide distances based on the reference structure, that follow the same sequence length distribution as TBIA-induced contacts, are shown in gray. D, the Kolmogorov-Smirnov metric, quantifies separation between two distributions on a 0 to 1 scale with 0 indicating no separation and 1 indicating complete separation; all D values correspond to p-values ≤ 10^–5.

Figure 5:. SHAPE-JuMP interactions detected for the RNase P RNA in the presence and absence of Mg²⁺.

(A) SHAPE-JuMP interactions for the most frequent 3% of deletions for RNase P in the presence (left) and absence (right) of Mg²⁺. Experimentally measured distances are shown with black lines; gray histograms represent distances of all nucleotide pairs in reference structure. (B) Internucleotide crosslinks, detected as cDNA deletions, superimposed on the secondary structure, colored by through-space distance as calculated from the reference structure. Nucleotides not visualized in the reference structure are shown within gray circles.

Figure 6:. Visualization of SHAPE-JuMP interactions on complex RNA structures.

Through-space interactions, detected as cDNA deletions, superimposed on secondary and tertiary structure models of (A) P546 intron domain (PDB ID 1gid), (B) VS ribozyme (4r4p), (C) RNase P catalytic domain (3dhs), and (D) group II intron (3igi). Internucleotide interactions are shown as lines, colored by through-space distance. Interactions are shown for most frequent 3% of measured deletions. Nucleotides not visualized in three-dimensional structure models are denoted with gray circles.

Figure 7:. SHAPE-JuMP directed structure refinement.

(A-C) Restraints superimposed on secondary structure and resulting three-dimensional models for the stepwise DMD refinement of the P546 intron domain. Five modeled structures (transparent red), consisting of the centroid and four models with lowest RMSD as compared to this centroid, aligned to the reference structure (gray) are shown. Restraints were added stepwise, (A) starting with the base paired secondary structure, (B) adding SHAPE-JuMP restraints at multi-helix junctions (orange lines), and (C) adding high frequency proximity interactions (purple lines). Lengths of restraint wells used during DMD refinement are color-coded. (D-G) Structures obtained using JuMP data-informed DMD aligned to the (D) P546 domain (PDB ID 1gid), (E) VS ribozyme (4r4p), (F) RNase P catalytic domain (3dhs), and (G) group II intron (3igi). JuMP restraints were mapped on to final models. The five models with the shortest restraint distance ranges were taken as representative of the simulation. Structures are colored by major helical elements. Modeled and accepted structures are shown with transparent and solid backbone traces, respectively. RMSD values are shown for: models with shortest restraint distance range, centroid of the largest cluster, and lowest RMSD model obtained. RMSD₁₀₀ values (in parentheses) report a length normalized RMSD. Regions not visualized in accepted structures are indicated with small spheres.