mRNA structure is a key determinant of translation efficiency for individual transcripts, yet its role in coordinating complex physiological processes remains elusive. We profiled mRNA structures during yeast meiosis, deriving a high-resolution structurome covering ~70% of annotated mRNAs, with multi-time-point measurements for 2,084 mRNAs. Transcripts upregulated during meiosis generally have flexible structures that enhance translation. In contrast, complex structures impede translation during meiosis by reducing ribosome flux in coding regions and promoting alternative initiation, including in the highly abundant transcript CCW22. We uncovered a high-low-high oscillation in cytoplasmic RNA helicase levels, which dynamically reprograms cell-wide translational preferences for RNA structure, shaping the temporal translation of hundreds of mRNAs. Disrupting RNA structure in CCW22 or altering Ded1p helicase levels interfered with meiotic proteostasis, hindering meiosis progression. Our study reveals that the concerted action of RNA structure and RNA helicases coordinates cell-wide translation dynamics, highlighting a potent post-transcriptional regulatory layer during meiosis when transcription is limited by chromosome condensation.

Structures in the 5 ' untranslated regions (UTRs) of mRNAs can physically modulate translation efficiency by impeding the scanning ribosome or by sequestering the translational start site. We assessed the impact of stable protein binding in 5 '- and 3 '-UTRs on translation efficiency by targeting the MS2 coat protein to a reporter RNA via its hairpin recognition site. Translation was assessed from the reporter RNA when coexpressed with MS2 coat proteins of varying affinities for the RNA, and at different expression levels. Binding of high-affinity proteins in the 5 '-UTR hindered translation, whereas no effect was observed when the coat protein was targeted to the 3 '-UTR. Inhibition of translation increased with coat protein concentration and affinity, reaching a maximum of 50%-70%. MS2 proteins engineered to bind two reporter mRNA sites had a stronger effect than those binding a single site. Our findings demonstrate that protein binding in an mRNA 5 '-UTR physically impedes translation, with the effect governed by affinity, concentration, and sterics.

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to nonbridging phosphate oxygens, creating a localized region of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

Extensive RNA-protein interactions occur throughout the lifecycle of an mRNA that up- and down-regulate mRNA translation and degradation. Modulating interactions between regulatory proteins and mRNAs can have large effects on gene expression and might be useful for creating therapeutic manipulations, especially for difficult-to-drug proteins. Here, we directed three degradation-inducing proteins that normally bind the 5' cap, 3'-untranslated region (UTR), or 3' poly(A) tail to unconventional sites spanning coding and noncoding regions across a reporter mRNA. DCP2, the 5' decapping enzyme, reduced expression only when targeted to the 5'-UTR. ZFP36L2, a 3'-UTR adapter protein, reduced expression when directed to either the 3'-UTR or the 3' half of the coding sequence, the latter region outside its conventional site. CNOT7, a catalytic subunit within the CCR4-NOT deadenylase complex, reduced expression when directed anywhere in the mRNA, most strongly at both "ends" in the 5'- and 3'-UTRs. mRNAs can therefore be degraded by directing proteins to positions far from their conventionally understood regulatory sites. Our study reveals extensive through-space functional proximity across an mRNA. These observations have broad implications for understanding large-scale RNA structure and for emerging applications in therapeutically targeted RNA degradation.

RNAs are critical regulators of gene expression, and their functions are often mediated by complex secondary and tertiary structures. Structured regions in RNA can selectively interact with small molecules-via well-defined ligand-binding pockets-to modulate the regulatory repertoire of an RNA. The broad potential to modulate biological function intentionally via RNA-ligand interactions remains unrealized, however, due to challenges in identifying compact RNA motifs with the ability to bind ligands with good physicochemical properties (often termed drug-like). Here, we devise fpocketR, a computational strategy that accurately detects pockets capable of binding drug-like ligands in RNA structures. Remarkably few, roughly 50, of such pockets have ever been visualized. We experimentally confirmed the ligandability of novel pockets detected with fpocketR using a fragment-based approach introduced here, Frag-MaP, that detects ligand-binding sites in cells. Analysis of pockets detected by fpocketR and validated by Frag-MaP reveals dozens of sites able to bind drug-like ligands, supports a model for RNA secondary structural motifs able to bind quality ligands, and creates a broad framework for understanding the RNA ligand-ome.

Small molecules that bind specific sites in RNAs hold promise for altering RNA function, manipulating gene expression, and expanding the scope of druggable targets beyond proteins. Identifying binding sites in RNA that can engage ligands with good physicochemical properties remains a significant challenge. fpocketR is a software and framework for identifying, characterizing, and visualizing ligand-binding sites in RNA. fpocketR was optimized, through a comprehensive analysis of currently available RNA-ligand complexes, to identify pockets in RNAs able to bind small molecules possessing favorable properties, generally termed drug-like. Here, we demonstrate multiple, complex, uses of fpocketR to analyze RNA-ligand interactions and novel pockets in small and large RNAs, to assess ensembles of RNA structure models, to identify pockets in dynamic RNA systems, and to evaluate the shapes of RNA pockets. fpocketR performs best with RNA structures visualized at atomistic resolution but also provides useful information with lower resolution structures and computational models. fpocketR is a powerful, ligand-agnostic tool for discovery and analysis of targetable pockets in RNA molecules.

A tumor-suppressive long noncoding RNA (lncRNA) DRAIC ( d own-regulated RN A in c ancers) inhibits NF-κB activity and physically interacts with IKKα, a kinase component of the IKK complex, in several cancer types. Here we explore the precise molecular mechanism involved in this interaction and suppression. Using SHAPE-MaP, we identified a 36-nucleotide hairpin (A+B ) within DRAIC that is necessary and sufficient for its anti-oncogenic function. RNA immunoprecipitation (RIP) and Electrophoretic mobility shift assays (EMSA) confirmed this hairpin physically interacts with the coiled coil domain of IKKα. A+B RNA has a high binding affinity (KD ∼1-7 nM) to the coiled-coil domain of IKKα. The binding of A+B disrupts the dimerization of NEMO and IKKα coiled-coil domains, a critical step for IKK action. Consistent with this, A+B inhibits the phosphorylation of the NF-κB inhibitor IκBα and suppresses NF-κB activity. Publicly available tumor RNAseq data revealed that alternative splicing modulates the presence of this critical hairpin: the inclusion of exon 4a (encoding one side of the A+B hairpin) in lung tumors correlates with reduced NF-κB activity. By demonstrating that the A+B hairpin is both necessary and sufficient to inhibit IKK and oncogenic phenotypes, this study underscores the centrality of IKKα interaction and NF-κB inhibition in DRAIC-mediated cancer suppression and indicates that the activity of this lncRNA is regulated by alternative splicing. This study also reveals the first example of a short RNA disrupting coiled-coil dimerization, offering a new approach to disrupt such dimerization in cancer biology.

Biogenesis of circular RNA usually involves a backsplicing reaction where the downstream donor site is ligated to the upstream acceptor site by the spliceosome. For this reaction to occur, these sites must be in proximity. Inverted repeat sequences, such as Alu elements, if positioned in the upstream and downstream introns, can base pair and represent one mechanism for inducing proximity. Here, we investigate the pre-mRNA structure of the human HIPK3 gene at exon 2, which forms a circular RNA via backsplicing. We leverage multiple chemical probing approaches, including the recently developed SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) strategy, to characterize secondary and tertiary interactions in the pre-mRNA that govern backsplicing. Our data confirm that the antisense Alu elements AluSz(-) and AluSq2(+), in the upstream and downstream introns, form a highly paired interaction. Circularization requires formation of long-range Alu-mediated base pairs but does not require the full-length AluSq2(+). In addition to confirming long-range base pairs, our SHAPE-JuMP data identified multiple long-range interactions between non-pairing nucleotides. Genome-wide analysis of inverted repeats flanking circular RNAs confirms that the presence of these elements favors circularization, but with modest predictive power. Together, our study suggests that secondary structure considerations alone do not fully explain backsplicing and that additional interactions are involved.

RNA-based fluorogenic aptamers, such as Mango, are uniquely powerful tools for imaging RNA that activate the fluorescence of a weakly or non-fluorescent small molecule when bound. A central challenge has been to develop brighter, more specific and high-affinity aptamer–ligand systems for cellular imaging. Here we report an ultrabright fluorophore for the Mango II system discovered using a structure-informed, fragment-based small-molecule microarray approach. This dye—termed SALAD1 (structure-informed, array-enabled LigAnD 1)—exhibits subnanomolar aptamer affinity and 3.5-fold brighter fluorescence than Mango II-TO1–biotin pair, a widely used fluorogenic system. Performance was improved by modulating RNA-dye molecular recognition without altering the fluorophore’s π-system. High-resolution X-ray structures reveal the binding mode for SALAD1, which exhibits improved pocket occupancy, a more defined binding pose and a unique bonding interaction with potassium. SALAD1 is cell-permeable and facilitates improved in-cell confocal RNA imaging. This work introduces an additional RNA-activated fluorophore demonstrating how fragment-based ligand discovery can be used to create high-performance ligands for RNA targets. Fluorogenic RNA aptamers such as Mango are powerful tools for RNA imaging. An ultrabright RNA-activated fluorophore with improved photophysical performance has now been developed using a structure-informed, fragment-based screening approach. This system was studied using high-resolution crystallography and applied to image RNA in cells.

Abstract Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-nt raiA bacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure of raiA at 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.

In the first of many thematic issues marking the 30th anniversary of Cell Chemical Biology, we highlight the contribution of chemical biology to RNA biology in a special issue on RNA modulation. We asked several leaders in the field to share their opinions on the current challenges and opportunities in RNA biology.

Splicing factors are affected by recurrent somatic mutations and copy number variations in several types of haematologic and solid malignancies, which is often seen as prima facie evidence that splicing aberrations can drive cancer initiation and progression. However, numerous spliceosome components also 'moonlight' in DNA repair and other cellular processes, making their precise role in cancer difficult to pinpoint. Still, few would deny that dysregulated mRNA splicing is a pervasive feature of most cancers. Correctly interpreting these molecular fingerprints can reveal novel tumour vulnerabilities and untapped therapeutic opportunities. Yet multiple technological challenges, lingering misconceptions, and outstanding questions hinder clinical translation. To start with, the general landscape of splicing aberrations in cancer is not well defined, due to limitations of short-read RNA sequencing not adept at resolving complete mRNA isoforms, as well as the shallow read depth inherent in long-read RNA-sequencing, especially at single-cell level. Although individual cancer-associated isoforms are known to contribute to cancer progression, widespread splicing alterations could be an equally important and, perhaps, more readily actionable feature of human cancers. This is to say that in addition to 'repairing' mis-spliced transcripts, possible therapeutic avenues include exacerbating splicing aberration with small-molecule spliceosome inhibitors, targeting recurrent splicing aberrations with synthetic lethal approaches, and training the immune system to recognize splicing-derived neoantigens.

A powerful approach for studying the functional consequences of site-specific RNA-protein interactions is to artificially tether a protein to a messenger (or noncoding) RNA through a selective, high-affinity interaction. We share a strategy for evaluating the contribution of protein positioning within an mRNA on gene expression. We introduced an RNA hairpin recognition site for the MS2 coat protein into the untranslated regions or coding sequence of mRNAs expressing a luminescent reporter protein, NanoLuc. Effector proteins fused to the MS2 coat protein could thus be targeted to distinct regions across the mRNA. We illustrate this approach using ZFP36L2, which recruits the CCR4-NOT complex for poly(A) tail deadenylation. Tethering ZFP36L2 to the 3'-UTR decreased NanoLuc expression, as expected, given the known interaction of this adapter protein with adenine uridine-rich elements (AREs). Intriguingly, ZFP36L2 also decreased NanoLuc expression when bound within the coding sequence, revealing that ZFP36L2-and potentially many other mRNA regulatory proteins-can function when targeted to diverse locations within an mRNA. This multi-target tethering strategy enables exploration of the interplay between mRNA-protein proximity and gene expression.

Chemical probing technologies enable high-throughput examination of diverse structural features of RNA including local nucleotide flexibility, RNA secondary structure, protein- and ligand-binding, through-space interaction networks, and multi-state structural ensembles. Performing these experiments, by themselves, does not directly lead to biological insight. Instead, deep understanding of RNA structure-function relationships typically requires evaluating a system under structure- and function-altering conditions, linking these data with additional information, and visualizing multi-layered relationships. Current platforms lack the broad accessibility, flexibility, and efficiency needed to iterate on integrative analyses of these diverse, complex data. Here, we share the RNA visualization and graphical analysis toolset RNAvigate, a straightforward and flexible Python library. RNAvigate currently automatically parses twenty-one standard file formats (primary sequence annotations, per- and inter-nucleotide data, and secondary and tertiary structures) and outputs eighteen plot types. These features enable efficient exploration of nuanced relationships between chemical probing data, RNA structure, and motif annotations across multiple experimental samples. Compatibility with Jupyter Notebooks enables non-burdensome, reproducible, transparent and organized sharing of multi-step analyses and data visualization strategies. RNAvigate simplifies examination of multi-layered RNA structure information and accelerates discovery and characterization of RNA-centric functions in biology.

Abstract Our group recently discovered that the circular RNA (circRNA), CDR1as, promotes lung cancer metastasis in part through the stabilization of the coding gene, CDR1. The purpose of this study was to identify intramolecular RNA secondary structures of CDR1as and determine whether there is a functional relationship between the CDR1as secondary structure and non-small cell lung cancer (NSCLC) metastasis. We employed the chemical probing approach selective 2’- hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to experimentally inform structure predictions of CDR1as. The SHAPE-MaP profiling experiments revealed that CDR1as is remarkably unstructured overall, with an exception of a highly probable and consistent stem-loop structure embedding the CDR1as backsplice junction (BSJ). Additionally, BSJ-embedding secondary structures were identified in all other mammalian circRNAs studied in a similar chemical probing experiment. To evaluate the role of the BSJ structure on CDR1as expression, we sterically disrupted the BSJ structure using a chemically modified, inert antisense oligonucleotide (BSJ-ASO) lacking a gapmer in order to block RNAse H activity. After transfection of the structure disrupting BSJ-ASO into several cell lines, we observed a 90-95% reduction of CDR1as expression by the BSJ-ASO by 24 hours. Furthermore, the BSJ-ASO significantly inhibited cancer cell proliferation and spheroid formation in several NSCLC models. Strikingly, using RNA stability assays we observed that the BSJ-ASO caused dramatic reductions in CDR1as levels within 5 minutes of transfection, suggesting a possibly hydrolytic process. A functional screen of silencing curated CDR1as RNA-binding proteins failed to rescue the loss of CDR1as expression following BSJ-ASO treatment. We next tested whether an RNA-catalyzed mechanism of action is possible. By incubating total RNA with the BSJ-ASO in buffers of varying magnesium chloride concentrations in vitro, we observed a dramatic decrease in CDR1as levels similar to that of the BSJ-ASO cellular transfection experiments, suggestive of ribozyme-like activity. Interestingly, use of a primer mapping approach indicated two putative cut sites within CDR1as. Further chemical probing following treatment with the BSJ-ASO revealed a new, highly probable secondary structure upstream of the BSJ, which appears similar to a hairpin ribozyme when modeled two-dimensionally. Taken together, our work reveals that intramolecular RNA secondary structures can have critical roles in maintaining circRNA cellular stability and function, and strategic steric disruption of these structures may induce circRNA self-cleavage. Citation Format: Aaron C Chack, Emily B Harrison, Caroline J Aufgebauer, Mark A Boerneke, Patrick S Irving, Edgar M Faison, Jingyu Zhao, Qi Zhang, Kevin M Weeks, Chad V Pecot. RNA secondary structures mediate circular RNA stability and function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A023.

RNA molecules fold to form complex internal structures. Many of these RNA structures populate ensembles with rheostat-like properties, with each state having a distinct function. Until recently, analysis of RNA structures, especially within cells, was limited to modeling either a single averaged structure or computationally-modeled ensembles. These approaches obscure the intrinsic heterogeneity of many structured RNAs. Single-molecule correlated chemical probing (smCCP) strategies are now making it possible to measure and deconvolute RNA structure ensembles based on efficiently executed chemical probing experiments. Here, we provide an overview of fundamental single-molecule probing principles, review current ensemble deconvolution strategies, and discuss recent applications to diverse biological systems. smCCP is enabling a revolution in understanding how the plasticity of RNA structure is exploited in biological systems to respond to stimuli and alter gene function. The energetics of RNA ensembles are often subtle and a subset can likely be targeted to modulate disease-associated biological processes.

Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, information about base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here, we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified an smCCP fingerprint that is selective for states containing a tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have a higher-order structure. These studies pave the way for the use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites(1-4). However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.

ABSTRACT To survive stress, eukaryotes selectively translate stress-related transcripts while inhibiting growth-associated protein production. How this translational reprogramming occurs under biotic stress has not been systematically studied. To identify common features shared by transcripts with stress-upregulated translation efficiency (TE-up), we first performed high-resolution ribosome-sequencing in Arabidopsis during pattern-triggered immunity and found that TE-up transcripts are enriched with upstream open reading frames (uORFs). Under non-stress conditions, start codons of these uORFs (uAUGs) have higher-than-background ribosomal association. Upon immune induction, there is an overall downshift in ribosome occupancy at uAUGs, accompanied by enhanced translation of main ORFs (mORFs). Using in planta nucleotide-resolution mRNA structurome probing, we discovered that this stress-induced switch in translation is mediated by highly structured regions detected downstream of uAUGs in TE-up transcripts. Without stress, these structures are responsible for uORF-mediated inhibition of mORF translation by slowing progression of the translation preinitiation complex to initiate translation from uAUGs, instead of mAUGs. In response to immune induction, uORF-inhibition is alleviated by three Ded1p/DDX3X-homologous RNA helicases which unwind the RNA structures, allowing ribosomes to bypass the inhibitory uORFs and upregulate defence protein production. Conservation of the RNA helicases suggests that mRNA structurome remodelling is a general mechanism for stress-induced translation across kingdoms.