A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE Chemistry

Mortimer, Stefanie; Weeks, Kevin M.

doi:10.1021/ja0704028

Cited by 328 publications

(433 citation statements)

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“…hSHAPE experiments [64–66] were performed on in vitro transcribed RNAs corresponding to nucleotides 1 to 713 using benzoyl cyanide (BzCN) and following the protocol described previously [25,33,57]. Briefly, RNAs were modified in a buffer favoring gRNA dimerization (50 mM sodium cacodylate (pH 7.5), 300 mM KCl and 5 mM MgCl 2 ) in the presence of 2 µg total yeast tRNA (Sigma Aldrich).…”

Section: Methodsmentioning

confidence: 99%

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

et al. 2018

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Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

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Section: Methodsmentioning

confidence: 99%

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

et al. 2018

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“…All RNAs were synthesized by in vitro transcription using PCR-generated templates (28). RNAs analyzed by SHAPE and hydroxyl radical footprinting were embedded in 5Ј and 3Ј structure cassette sequences (34).…”

Section: Methodsmentioning

confidence: 99%

“…The general procedure was that outlined previously (28). Briefly, fluorescently labeled DNA primers were annealed to the RNA (10 L, from the previous modification steps) and extended by reverse transcription.…”

Section: Shape Analysismentioning

confidence: 99%

“…Briefly, fluorescently labeled DNA primers were annealed to the RNA (10 L, from the previous modification steps) and extended by reverse transcription. cDNA extension products, along with dideoxy sequencing markers (28), were separated by capillary electrophoresis using an Applied Biosystems 3130 instrument. Raw traces were processed using ShapeFinder (36).…”

Section: Shape Analysismentioning

confidence: 99%

“…This strong differential reactivity with the slowly reacting reagent indicates that this C2Ј-endo nucleotide undergoes an extraordinarily slow conformational change to reach the state in which the 2Ј-hydroxyl is reactive toward acylation (t 1/2 ϭ 10-100 s) (17). When a similar experiment is performed in the absence of Mg 2ϩ , where the RNA does not form a well-defined tertiary structure (20,28), the fast and slow SHAPE reagents show equivalent reactivities at A130 (red bar in bottom panel, Fig. 1B).…”

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confidence: 96%

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C2′-endo nucleotides as molecular timers suggested by the folding of an RNA domain

Mortimer

Weeks

2009

Proc. Natl. Acad. Sci. U.S.A.

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A striking and widespread observation is that higher-order folding for many RNAs is very slow, often requiring minutes. In some cases, slow folding reflects the need to disrupt stable, but incorrect, interactions. However, a molecular explanation for slow folding in most RNAs is unknown. The specificity domain of the Bacillus subtilis RNase P ribozyme undergoes a rate-limiting folding step on the minute time-scale. This RNA also contains a C2 -endo nucleotide at A130 that exhibits extremely slow local conformational dynamics. This nucleotide is evolutionarily conserved and essential for tRNA recognition by RNase P. Here we show that deleting this single nucleotide accelerates folding by an order of magnitude even though this mutation does not change the global fold of the RNA. These results demonstrate that formation of a single stacking interaction at a C2 -endo nucleotide comprises the rate-determining step for folding an entire 154 nucleotide RNA. C2 -endo nucleotides exhibit slow local dynamics in structures spanning isolated helices to complex tertiary interactions. Because the motif is both simple and ubiquitous, C2 -endo nucleotides may function as molecular timers in many RNA folding and ligand recognition reactions.RNA folding ͉ RNA SHAPE chemistry T o function properly inside the cell, RNA molecules undergo complex folding transitions to form specific, biologically active, three-dimensional structures (1). These essential structures involve both local base pairing and also complex higherorder tertiary interactions. A persistent and incompletely explained observation is that many RNAs fold very slowly, on timescales requiring minutes or longer. RNAs that fold slowly span all sizes including small riboswitch and catalytic RNAs, medium-sized catalytic introns, and the large RNAs in ribosomes (2-6).Slow folding has important consequences both because correct folding ultimately governs the rate at which an RNA can perform its biological function and because bottlenecks in assembly potentially require cellular mechanisms to overcome slow folding. In some cases, slow folding results from formation of stable, non-native, and kinetically trapped states (7,8). Such misfolding is remedied, in part, by cellular chaperone activities (9-11). In many cases, the molecular basis for slow folding is unknown.The intricate three-dimensional structures formed by RNA molecules ultimately reflect the underlying contributions of individual nucleotides. Most nucleotides in an RNA molecule exist in the C3Ј-endo conformation. Although less frequent, the C2Ј-endo conformation is highly overrepresented in catalytic active sites and in critical tertiary structures (12, 13). At the static structure level, the C2Ј-endo conformation induces greater helical twist in an A-form helix (14, 15) and spans a much longer 5Ј-to-3Ј distance (12,16). At the level of local motion, some C2Ј-endo nucleotides exhibit extraordinarily slow conformational dynamics with half-lives on the 10-100-s timescale (17).Here we show that the slow conformational ...

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SHAPE–Seq: High‐Throughput RNA Structure Analysis

Mortimer

Trapnell

Aviran

et al. 2012

CP Chemical Biology

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Knowledge of RNA structure is critical to understanding both the important functional roles of RNA in biology and the engineering of RNA to control biological systems. This article contains a protocol for selective 2′‐hydroxyl acylation analyzed by primer extension and sequencing (SHAPE‐Seq) that, through a combination of structure‐dependent chemical probing and next‐generation sequencing technologies, achieves structural characterization of hundreds of RNAs in a single experiment. This protocol is applicable in a variety of conditions, and represents an important tool for understanding RNA biology. The protocol includes methods for the design and synthesis of RNA mixtures for study, and the construction and analysis of structure‐dependent sequencing libraries that reveal structural information of the RNAs in the mixtures. The methods are generally applicable to studying RNA structure and interactions in vitro in a variety of conditions, and allows for the rapid characterization of RNA structures in a high‐throughput manner. Curr. Protoc. Chem. Biol. 4:275‐297 © 2012 by John Wiley & Sons, Inc.

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A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE Chemistry

Cited by 328 publications

References 12 publications

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

C2′-endo nucleotides as molecular timers suggested by the folding of an RNA domain

SHAPE–Seq: High‐Throughput RNA Structure Analysis

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