Probing the dynamic RNA structurome and its functions

Abstract

RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome -the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.Sections glyoxal and other α-ketoaldehydes, which react with unpaired guanines 14 , and carbodiimide reagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which display selectivity for unpaired guanines and uracils, or for G:U wobble pairs 15,16 .By contrast, chemical probing by selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) measures the flexibility of the RNA backbone 17 , which is generally used as a proxy for base-pairing: when the SHAPE reagent forms an adduct with the 2′-OH of a structurally flexible nucleotide, the position is assumed to be unpaired. Moreover, specific structural states have been shown to promote SHAPE Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author selfarchiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Chemical RNA Cross-Linking: Mechanisms, Computational Analysis, and Biological Applications

“…While conventional physical methods, such as NMR, X-ray crystallography and cryo-EM have been instrumental in protein structure analysis, their applications in RNA have been more limited, primarily due to RNA’s large size, high flexibility, and strong dependence on physiological environments. , Computational structure modeling based on minimal free energy calculations and phylogenetic analysis of conservation and covariation also suffer from multiple limitations, such as lack of understanding of RNA folding rules and high computational cost. Therefore, direct measurements of RNA structures in cells have been critical for understanding RNA behavior in various biological and pathological processes . A variety of chemical reactions have been developed and exploited that can modify RNA at certain positions (Figure ) depending on nucleotide reactivity, flexibility, or accessibility, which correlate with RNA structural constraints .…”

“…Therefore, direct measurements of RNA structures in cells have been critical for understanding RNA behavior in various biological and pathological processes. 13 A variety of chemical reactions have been developed and exploited that can modify RNA at certain positions ( Figure 1 ) depending on nucleotide reactivity, flexibility, or accessibility, which correlate with RNA structural constraints. 14 For example, dimethyl sulfate (DMS) selectively alkylates the N1 position of adenine and N3 position of cytosine on unpaired nucleotides 15 and the 2′-OH in flexible regions can be acylated with Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) reagents.…”

Chemical RNA Crosslinking: Mechanisms, Computational Analysis and Biological Applications

“…Therefore, direct measurements of RNA structures in cells have been critical for understanding RNA behavior in various biological and pathological processes. 13 A variety of chemical reactions have been developed and exploited that can modify RNA at certain positions (Fig. 1) depending on nucleotide reactivity, flexibility, or accessibility, which correlate with RNA structural constraints.…”

Contemporary Progress and Opportunities in RNA-Targeted Drug Discovery

“…Indeed, recent studies have shown that cellular RNAs are invariably bound to and often modified by proteins. , Yet, how this regulation, as well as other aspects of the cellular environment, affects RNA structural ensembles, and in turn ligandability, is almost completely unknown. Fortunately, new technologies, most notably high-throughput sequencing-based RNA structure mapping methods, are enabling us to begin to characterize the RNA structurome in living cells . These efforts are likely to reveal new insights into the complexity of RNA folding and structural ensembles, improving our chances of identifying structured RNA targets amenable for selective small molecule targeting in vivo , as well as their bioactive RNA conformations .…”

“…Fortunately, new technologies, most notably high-throughput sequencing-based RNA structure mapping methods, are enabling us to begin to characterize the RNA structurome in living cells. 111 These efforts are likely to reveal new insights into the complexity of RNA folding and structural ensembles, improving our chances of identifying structured RNA targets amenable for selective small molecule targeting in vivo, as well as their bioactive RNA conformations. 111 In tandem, new technologies are also being developed and deployed for the large-scale cataloguing of RNA−protein interactions, 112 as well as a "call-to-arms" for the development of methods capable of reading out RNA modifications, 113 which at present are poorly understood.…”