High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly

Abstract

RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and non-additive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes.

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

“…1B) that can be visualized by high-throughput capillary electrophoresis ( Fig. 1C; Wilkinson et al 2008;McGinnis et al 2009;Watts et al 2009). SHAPE reactivities correlate strongly with model-free measurements of molecular order and are largely independent of nucleotide type or solvent accessibility (Gherghe et al 2008;Wilkinson et al 2009;McGinnis et al 2012).…”

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

“…20 In all cases the background values observed in the NMIA (-) RNA were subtracted from the NMIAtreated RNA prior to SHAPE data normalization. 39 The mean SHAPE reactivity values obtained for free RNA and IRAB-treated RNA (20 mM) is shown in Figure 3A. Nucleotides with normalized reactivity >0.5 (red shaded) are considered to be highly flexible, likely unpaired.…”

“…To quantify the effect of IRAB using an unbiased approach for visualizing the compound-induced modification of the IRES local flexibility, we obtained the difference profile 39,41 by subtracting the reactivity of the free RNA from that of the IRAB treated RNA (Fig. 4A).…”

“…All reactivity values were normalized to this average value to generate the corresponding SHAPE reactivity profiles. 39 Data from 3 independent assays were used to calculate the mean ( §SD) SHAPE reactivity. Differences in SHAPE reactivity induced by incubation of the IRES with IRAB were analyzed by unpaired two-tail Student´s t-test; differences were considered significant when P < 0.05.…”

“…Fe(II)-EDTA [7.5 mM Fe(SO 4 ) 2 (NH 4 ) 2 ¢6H 2 O and 11.25 mM EDTA, pH 8.0], 0.3% hydrogen peroxide and 150 mM sodium ascorbate solutions were freshly prepared before each experiment. 39 Hydroxyl radical cleavage was initiated by adding 1 ml of Fe(II)-EDTA complex, 1 ml of sodium ascorbate and 1 ml of hydrogen peroxide solution to the reaction. A control reaction lacking Fe(II)-EDTA was performed in parallel.…”