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.
Mutational profiling (MaP) enables detection of sites of chemical modification in RNA as sequence changes during reverse transcription (RT), subsequently read out by massively parallel sequencing. We ...introduce ShapeMapper 2, which integrates careful handling of all classes of adduct-induced sequence changes, sequence variant correction, basecall quality filters, and quality-control warnings to now identify RNA adduct sites as accurately as achieved by careful manual analysis of electrophoresis data, the prior highest-accuracy standard. MaP and ShapeMapper 2 provide a robust, experimentally concise, and accurate approach for reading out nucleic acid chemical probing experiments.
RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is ...unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.
RNA is arguably the most versatile biological macromolecule because of its ability both to encode and to manipulate genetic information. The diverse roles of RNA depend on its ability to fold back on ...itself to form biologically functional structures that bind small molecule and large protein ligands, to change conformation, and to affect the cellular regulatory state. These features of RNA biology can be structurally interrogated using chemical mapping experiments. The usefulness and applications of RNA chemical probing technologies have expanded dramatically over the past five years because of several critical advances. These innovations include new sequence-independent RNA chemistries, algorithmic tools for high-throughput analysis of complex data sets composed of thousands of measurements, new approaches for interpreting chemical probing data for both secondary and tertiary structure prediction, facile methods for following time-dependent processes, and the willingness of individual research groups to tackle increasingly bold problems in RNA structural biology.
RNA molecules are essential for cellular information transfer and gene regulation, and RNAs have been implicated in many human diseases. Messenger and non-coding RNAs contain highly structured ...elements, and evidence suggests that many of these structures are important for function. Targeting these RNAs with small molecules offers opportunities to therapeutically modulate numerous cellular processes, including those linked to 'undruggable' protein targets. Despite this promise, there is currently only a single class of human-designed small molecules that target RNA used clinically - the linezolid antibiotics. However, a growing number of small-molecule RNA ligands are being identified, leading to burgeoning interest in the field. Here, we discuss principles for discovering small-molecule drugs that target RNA and argue that the overarching challenge is to identify appropriate target structures - namely, in disease-causing RNAs that have high information content and, consequently, appropriate ligand-binding pockets. If focus is placed on such druggable binding sites in RNA, extensive knowledge of the typical physicochemical properties of drug-like small molecules could then enable small-molecule drug discovery for RNA targets to become (only) roughly as difficult as for protein targets.
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at ...single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.
mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of ...mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.
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•E. coli mRNAs adopt highly diverse and complex structures•Translation is the main source of mRNA structural destabilization in cells•Translation efficiency is strongly correlated with ribosome-binding-site structure•Conserved structured elements found in 35% of UTRs
High-resolution probing of hundreds of genes in living E. coli cells reveals that bacterial mRNAs fold into highly diverse and complex structures and that these structures have widespread regulatory functions.
Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, which makes it difficult to understand how RNA structure governs function. Here we describe ...selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2'-hydroxyl acylation by SHAPE are encoded as noncomplementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure, and we used it to define a new model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary-structure models, enables analysis of low-abundance RNAs, disentangles sequence polymorphisms in single experiments and will ultimately democratize RNA-structure analysis.
Piercing the fog of the RNA structure-ome Weeks, Kevin M
Science (American Association for the Advancement of Science),
08/2021, Volume:
373, Issue:
6558
Journal Article
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA ...nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.
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