A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Marinus, Tycho; Fessler, Adam B.; Ogle, Craig A.; Incarnato, Danny

doi:10.1101/2020.08.31.274761

Cited by 8 publications

(11 citation statements)

References 38 publications

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“…Three broad classes of SHAPE reagents have been developed based on isatoic anhydride, 12,14,37 imidazolide, 38,39 and benzoyl cyanide 40 scaffolds (Figure 2E). The most important considerations for selecting a SHAPE reagent are half-life and solubility.…”

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confidence: 99%

SHAPE Directed Discovery of New Functions in Large RNAs

Weeks

2021

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations CONSPECTUS: RNA lies upstream of nearly all biology and functions as the central conduit of information exchange in all cells. RNA molecules encode information both in their primary sequences and in complex structures that form when an RNA folds back on itself. From the time of discovery of mRNA in the late 1950s until quite recently, we had only a rudimentary understanding of RNA structure across vast regions of most messenger and noncoding RNAs. This deficit is now rapidly being addressed, especially by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, mutational profiling (MaP), and closely related platform technologies that, collectively, create chemical microscopes for RNA. These technologies make it possible to interrogate RNA structure, quantitatively, at nucleotide resolution, and at large scales, for entire mRNAs, noncoding RNAs, and viral RNA genomes. By applying comprehensive structure probing to diverse problems, we and others are showing that control of biological function mediated by RNA structure is ubiquitous across prokaryotic and eukaryotic organisms. Work over the past decade using SHAPE-based analyses has clarified key principles. First, the method of RNA structure probing matters. SHAPE-MaP, with its direct and one-step readout that probes nearly every nucleotide by reaction at the 2′-hydroxyl, gives a more detailed and accurate readout than alternatives. Second, comprehensive chemical probing is essential. Focusing on fragments of large RNAs or using meta-gene or statistical analyses to compensate for sparse data sets misses critical features and often yields structure models with poor predictive power. Finally, every RNA has its own internal structural personality. There are myriad ways in which RNA structure modulates sequence accessibility, protein binding, translation, splice-site choice, phase separation, and other fundamental biological processes. In essentially every instance where we have applied rigorous and quantitative SHAPE technologies to study RNA structure−function interrelationships, new insights regarding biological regulatory mechanisms have emerged. RNA elements with more complex higher-order structures appear more likely to contain high-information-content clefts and pockets that bind small molecules, broadly informing a vigorous field of RNA-targeted drug discovery.The broad implications of this collective work are twofold. First, it is long past time to abandon depiction of large RNAs as simple noodle-like or gently flowing molecules. Instead, we need to emphasize that nearly all RNAs are punctuated with distinctive internal structures, a subset of which modulate function in profound ways. Second, structure probing should be an integral component of any effort that seeks to understand the functional nexuses and biological roles of large RNAs.

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confidence: 99%

SHAPE Directed Discovery of New Functions in Large RNAs

Weeks

2021

Acc. Chem. Res.

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“…The discovery that a blockbuster drug like Palbociclib binds to RNA with such potency and specificity demonstrates that drug-like molecules do indeed bind to and inhibit RNA, even in the absence of complex higher order RNA structures. Stem-loops distorted by internal loops and bulges, like HIV TAR, are the dominant secondary structure element in RNA and likely to form in cells where RNAs are less stably folded 14,12 , while more complex tertiary structures are unfolded by RNA-binding proteins, RNA helicases, and active translation. The or DMSO, depending on their solubility, then prepared to 100 µM in 490 µL in buffer (50 mM d19-deuterated bis-Tris buffer at pH 6.5 containing 11.1 µM sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSA) as chemical shift and intensity reference, all dissolved in 99.99% D2O).…”

Section: Discussionmentioning

confidence: 99%

“…Ribosomal RNA and riboswitches provide some of the best known examples of recognition of structured RNAs by small molecules 1,10 . However, the elaborate higher order folds observed in these RNAs are not common in non-coding RNAs and mRNAs, whose structures are frequently destabilized by single strand RNA binding proteins and during active translation 11,12 . Simpler secondary structures such as stem loops and internal loops are ubiquitous in cellular and viral RNAs and perform well-established regulatory functions by regulating pre-mRNA splicing, 3'-end processing, protein synthesis, etc.…”

Section: Introductionmentioning

confidence: 99%

The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

Shortridge

Vidalala

Varani

2022

Preprint

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The growing awareness of the role of RNA in human disease has motivated significant efforts to discover drug-like small molecules that target RNA. However, high throughput screening campaigns report very low hit rates and generally identify compounds with weak affinity, while most structures reported in Academic studies also lack the pharmacological properties of successful drugs. Even FDA-approved RNA-targeting drugs have only weak (10 uM) binding activity. Thus, it is often stated that only complex RNA structures, such as the ribosome or riboswitches, are amenable to small molecule chemistry. We report that the kinase inhibitor Palbociclib/Ibrance is a nM ligand for the HIV-1 TAR. It inhibits recruitment of the positive transcription elongation factor complex at nM concentrations and discriminates >20 fold. We further show that RNA binding can be fully decoupled from kinase inhibition, yielding a new molecule with even higher affinity for RNA. We thus demonstrate that nM affinity, specificity, and potent biochemical activity against 'undruggable' RNAs can be found in the chemical space of blockbuster drugs.

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“…SHAPE mapping using an appropriate electrophile to acylate the ribose 2'-hydroxyl groups efficiently interrogates the flexibility of local RNA structures in vitro and in vivo [24]. Several well-validated SHAPE reagents with different cell permeabilities, half-lives, and nucleotide biases are currently used for RNA structure mapping in cells [25][26][27]. In the case of Ty1 gRNA, all prior structural studies were performed using Nmethylisatoic anhydride (NMIA) [15,22,[28][29][30][31].…”

Section: Nmia Successfully Modifies Rna In the Yeast Cell Nucleusmentioning

confidence: 99%

Cell Compartment-specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome

Zawadzka¹,

Andrzejewska-Romanowska²,

Gumna³

et al. 2022

Preprint

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The structural transitions RNAs undergo during trafficking are not well-understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and synthesized in vitro, we detected Ty1 gRNA structural alternations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, while the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA:RNA interactions required for retrotransposition.

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A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Cited by 8 publications

References 38 publications

SHAPE Directed Discovery of New Functions in Large RNAs

SHAPE Directed Discovery of New Functions in Large RNAs

The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

Cell Compartment-specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome

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