Probing RNA Conformational Equilibria within the Functional Cellular Context

Abstract

Low-abundance short-lived non-native conformations referred to as excited states (ESs) are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs. We developed an approach for assessing the relative abundance of RNA ESs within the functional cellular context. Nuclear magnetic resonance (NMR) spectroscopy was used to estimate the degree to which substitution mutations bias conformational equilibria toward the inactive ES in vitro. The cellular activity of the ES-stabilizing mutants was used as an indirect measure of the conformational equilibria within the functional cellular context. Compensatory mutations that restore the ground-state conformation were used to control for changes in sequence. Using this approach, we show that the ESs of two regulatory RNAs from HIV-1, the transactivation response element (TAR) and the Rev response element (RRE), likely form in cells with abundances comparable to those measured in vitro, and their targeted stabilization may provide an avenue for developing anti-HIV therapeutics.

Keywords: HIV-1 transactivation response element; RNA drug discovery; RNA dynamics; RNA structure; RRE; Rev response element; TAR; cellular activity; conformational switches; excited state; structure mapping; transactivation.

Figure 1.

Evaluating the impact of the cellular environment on conformational equilibria using targeted stabilization of RNA ESs and functional readouts to measure the population of the active RNA GS. Substitution mutations (star) redistribute the WT RNA ensemble in vitro, increasing the abundance of an ES over the GS to various degrees measured by NMR spectroscopy. The impact of the mutations on cellular activity is then measured using an appropriate assay.

Figure 2.

Probing the conformational equilibrium of HIV-1 TAR in vitro and in vivo. (A) HIV-1 TAR RNA has two ESs that alter base pairing in the bulge (orange), upper stem (blue), and apical loop (green). (B) Example of the stabilize-and-rescue approach for trapping TAR ES2 using the ES-stabilizing mutant G28U (orange) and its corresponding rescue mutation C37A (blue). For A and B, weak base pairs (open dot) and non-Watson-Crick base pairs (red dot) are indicated. (C) Summary of TAR mutants with mutated base pairs highlighted (red box). The extent of stabilization was estimated based on NMR line broadening (Figures S2–S3). (++) minimal line broadening, (+) partial line broadening, and (−) extensive line broadening. (D) Tat-dependent trans-activation assay for TAR mutants determined to be primarily in the GS conformation (black labels) or ES conformation (orange labels) based on NMR. HeLa cells were transfected with pFLuc-TAR reporter plasmid and an RLuc internal control in the presence (+ Tat) or absence (- Tat) of a Tat expression plasmid. Reported values are the quotient of FLuc and RLuc activities with values normalized to WT TAR for every independent replicate reported as the mean ± SD (n=8). Statistical significance of the difference in +Tat and -Tat activity between wtTAR and each mutant was determined after log-transformation of the data and prior to normalization to WT TAR; P-values between TAR variants are shown (two-sided ANOVA). (E) In vitro binding assay between TAR mutants and a peptide of the Tat arginine rich motif. Reported values are the mean ± SD (n=2) of the K_a (1/K_d) values normalized to wtTAR.

Figure 3.

Probing the conformational equilibrium of HIV-1 RRE in vitro and in vivo. (A) HIV-1 RRE RNA has two ESs that reshuffle the non-canonical base pairs at the purine-rich region (blue), U72 (yellow), and A68 (green) bulge. (B) Example of the stabilize-and-rescue approach for trapping RRE ES2 using an ES-stabilizing mutant A68C (orange) and corresponding rescue mutations G50A and C69U (blue). For A and B, weak base pairs (open dot) and non-Watson-Crick base pairs (red dot) are indicated. (C) Summary of RRE mutants with mutated base pairs highlighted (red box). The extent of stabilization was estimated based on NMR line broadening (Figure S2). (++) minimal line broadening, (+) partial line broadening, and (−) extensive line broadening. (D) Rev-RRE dependent RNA export assays of RRE mutants determined to be primarily in the GS conformation (black labels) or ES conformation (orange labels) based on NMR. 293T cell were transfected with pFLuc-RRE reporter plasmid and an RLuc internal control in the presence (+ Rev) or absence (-Rev) of a Rev expression plasmid. Reported values are the quotient of FLuc and RLuc activities with values normalized to WT RRE for every independent replicate reported as the mean ± SD (n=3). MP-1 is the control plasmid (no RRE). Statistical significance of the difference in +Rev and -Rev activity between wtRRE and each mutant was determined after log-transformation of the data; P-values between RRE-variants are shown (two-sided ANOVA). (E) Cell-based trans-activation assays to detect Rev-RRE binding. Tat-Rev fusion protein dependent trans-activation assays of RRE and mutants. HeLa cells were transfected with pFLuc-TAR/RRE reporter plasmid and an RLuc internal control in the presence of (+ Tat/Rev) or (+Tat) expression plasmid. At 48 hr post transfection, cell lysates were tested for luciferase activity. Reported values are the quotient of FLuc and RLuc activities with values normalized to WT RREII for every independent replicate reported as the mean ± SD (n=3). P-values between RRE variants are shown (two-sided ANOVA).