RNA Structural Elements Research Articles

Dissecting the Conformational Heterogeneity of Stem-Loop Substructures of the Fifth Element in the 5'-Untranslated Region of SARS-CoV-2.

Throughout the family of coronaviruses, structured RNA elements within the 5' region of the genome are highly conserved. The fifth stem-loop element from SARS-CoV-2 (5_SL5) represents an example of an RNA structural element, repeatedly occurring in coronaviruses. It contains a conserved, repetitive fold within its substructures SL5a and SL5b. We herein report the detailed characterization of the structure and dynamics of elements SL5a and SL5b that are located immediately upstream of the SARS-CoV-2 ORF1a/b start codon. Exploiting the unique ability of solution NMR methods, we show that the structures of both apical loops are modulated by structural differences in the remote parts located in their stem regions. We further integrated our high-resolution models of SL5a/b into the context of full-length 5_SL5 structures by combining different structural biology methods. Finally, we evaluated the impact of the two most common VoC mutations within 5_SL5 with respect to individual base-pair stability.

Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Sub-genomic flaviviral RNA elements increase the stability and abundance of recombinant AAV vector transcripts.

Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.

Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement.

Diversity-generating retroelements (DGRs) create massive protein sequence variation in ecologically diverse microbes. Variation occurs during reverse transcription of a protein-encoding RNA template coupled to misincorporation at adenosines. In the prototypical Bordetella bacteriophage DGR, the template must be surrounded by upstream and downstream RNA segments for cDNA synthesis by the reverse transcriptase bRT and associated protein Avd. The function of the surrounding RNA was unknown. Cryo-EM revealed that this RNA enveloped bRT and lay over barrel-shaped Avd, forming an intimate ribonucleoprotein (RNP). An abundance of essential interactions between RNA structural elements and bRT-Avd precisely positioned an RNA homoduplex for initiation of cDNA synthesis by cis -priming. Our results explain how the surrounding RNA primes cDNA synthesis, promotes processivity, terminates polymerization, and strictly limits mutagenesis to select proteins through mechanisms that are likely conserved in DGRs from distant taxa.

Structure and function of type IV IRES in picornaviruses: a systematic review

The Picornaviridae is a family of icosahedral viruses with single-stranded, highly diverse positive-sense RNA genomes. Virions consist of a capsid, without envelope, surrounding a core of RNA genome. A typical genome of picornavirus harbors a well-conserved and highly structured RNA element known as the internal ribosome entry site (IRES), functionally essential for viral replication and protein translation. Based on differences in their structures and mechanisms of action, picornaviral IRESs have been categorized into five types: type I, II, III, IV, and V. Compared with the type IV IRES, the others not only are structurally complicated, but also involve multiple initiation factors for triggering protein translation. The type IV IRES, often referred to as hepatitis C virus (HCV)-like IRES due to its structural resemblance to the HCV IRES, exhibits a simpler and more compact structure than those of the other four. The increasing identification of picornaviruses with the type IV IRES suggests that this IRES type seems to reveal strong retention and adaptation in terms of viral evolution. Here, we systematically reviewed structural features and biological functions of the type IV IRES in picornaviruses. A comprehensive understanding of the roles of type IV IRESs will contribute to elucidating the replication mechanism and pathogenesis of picornaviruses.

NMR characterization of RNA binding property of the DEAD-box RNA helicase DDX3X and its implications for helicase activity

The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes.

Combinatorial transcriptomic and genetic dissection of insulin/IGF-1 signaling-regulated longevity in Caenorhabditis elegans.

Classical genetic analysis is invaluable for understanding the genetic interactions underlying specific phenotypes, but requires laborious and subjective experiments to characterize polygenic and quantitative traits. Contrarily, transcriptomic analysis enables the simultaneous and objective identification of multiple genes whose expression changes are associated with specific phenotypes. Here, we conducted transcriptomic analysis of genes crucial for longevity using datasets with daf-2/insulin/IGF-1 receptor mutant Caenorhabditis elegans. Our analysis unraveled multiple epistatic relationships at the transcriptomic level, in addition to verifying genetically established interactions. Our combinatorial analysis also revealed transcriptomic changes associated with longevity conferred by daf-2 mutations. In particular, we demonstrated that the extent of lifespan changes caused by various mutant alleles of the longevity transcription factor daf-16/FOXO matched their effects on transcriptomic changes in daf-2 mutants. We identified specific aging-regulating signaling pathways and subsets of structural and functional RNA elements altered by different genes in daf-2 mutants. Lastly, we elucidated the functional cooperation between several longevity regulators, based on the combination of transcriptomic and molecular genetic analysis. These data suggest that different biological processes coordinately exert their effects on longevity in biological networks. Together our work demonstrates the utility of transcriptomic dissection analysis for identifying important genetic interactions for physiological processes, including aging and longevity.

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Fluorescent Ligand Equilibrium Displacement: A High-Throughput Method for Identification of FMN Riboswitch-Binding Small Molecules.

Antibiotic resistance remains a pressing global concern, with most antibiotics targeting the bacterial ribosome or a limited range of proteins. One class of underexplored antibiotic targets is bacterial riboswitches, structured RNA elements that regulate key biosynthetic pathways by binding a specific ligand. We developed a methodology termed Fluorescent Ligand Equilibrium Displacement (FLED) to rapidly discover small molecules that bind the flavin mononucleotide (FMN) riboswitch. FLED leverages intrinsically fluorescent FMN and the quenching effect on RNA binding to create a label-free, in vitro method to identify compounds that can bind the apo population of riboswitch in a system at equilibrium. The response difference between known riboswitch ligands and controls demonstrates the robustness of the method for high-throughput screening. An existing drug discovery library that was screened using FLED resulted in a final hit rate of 0.67%. The concentration response of each hit was determined and revealed a variety of approximate effective concentration values. Our preliminary screening data support the use of FLED to identify small molecules for medicinal chemistry development as FMN riboswitch-targeted antibiotic compounds. This robust, label-free, and cell-free method offers a strong alternative to other riboswitch screening methods and can be adapted to a variety of laboratory setups.

Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.

Programmable modulation of ribosomal frameshifting by mRNA targeting CRISPR-Cas12a system

Minus 1 programmed ribosomal frameshifting (-1 PRF) is a conserved translational regulation event essential for critical biological processes, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Efficient trans-modulation of the structured RNA element crucial to -1 PRF will endow the therapeutic application. Here, we demonstrate that CRISPR RNA can stimulate efficient -1 PRF. Assembled CRISPR-Cas12a, but not CRISPR-Cas9, complex further enhances -1 PRF efficiency through its higher capacity to stall translating ribosomes. We additionally perform CRISPR-Cas12a targeting to impair the SARS-CoV-2 frameshifting pseudoknot structure via a focused screening. We demonstrate that targeting CRISPR-Cas12a results in more than 70% suppression of -1 PRF invitro and about 50% suppression in mammalian cells. Our results show the expanded function of the CRISPR-Cas12 system in modulating -1 PRF efficiency through stalling ribosomes and deforming frameshifting stimulatory signals, which could serve as a new strategy for future coronavirus pandemics.

A generalizable scaffold-based approach for structure determination of RNAs by cryo-EM.

Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their intrinsic low signal to noise ratio. Methods to help resolve small proteins have been applied but development of similar approaches to aid in structural determination of small, structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5-5.0 Å. While lacking the detail of true high-resolution maps, these maps are suitable for model building and preliminary structure determination. We demonstrate this method helped faithfully recover the structure of several RNA elements of known structure, and that it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a robust system to aid in RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution.

Riboswitches as therapeutic targets: promise of a new era of antibiotics

ABSTRACT Introduction The growth of antibiotic resistance among bacterial pathogens is an impending global threat that can only be averted through the development of novel antibacterial drugs. A promising answer could be the targeting of riboswitches, structured RNA elements found almost exclusively in bacteria. Areas covered This review examines the potential of riboswitches as novel antibacterial drug targets. The limited mechanisms of action of currently available antibiotics are summarized, followed by a delineation of the functional mechanisms of riboswitches. We then discuss the potential for developing novel approaches that target paradigmatic riboswitches in the context of their bacterial gene expression machinery. Expert opinion We highlight potential advantages of targeting riboswitches in their functional form, embedded within gene expression complexes critical for bacterial survival. We emphasize the benefits of this approach, including potentially higher species specificity and lower side effects.

The Highly Conserved Stem-Loop II Motif Is Dispensable for SARS-CoV-2.

The stem-loop II motif (s2m) is an RNA structural element that is found in the 3' untranslated region (UTR) of many RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over 25 years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3' UTR of wild-type and s2m deletion viruses using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) and dimethyl sulfate mutational profiling and sequencing (DMS-MaPseq). These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3'-UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2. IMPORTANCE RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), contain functional structures to support virus replication, translation, and evasion of the host antiviral immune response. The 3' untranslated region of early isolates of SARS-CoV-2 contained a stem-loop II motif (s2m), which is an RNA structural element that is found in many RNA viruses. This motif was discovered over 25 years ago, but its functional significance is unknown. We created SARS-CoV-2 with deletions or mutations of the s2m and determined the effect of these changes on viral growth in tissue culture and in rodent models of infection. Deletion or mutation of the s2m element had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also observed no impact of the deletion on other known RNA structures in the same region of the genome. These experiments demonstrate that s2m is dispensable for SARS-CoV-2.

Discovery of Small Molecules Targeting the Frameshifting Element RNA in SARS-CoV-2 Viral Genome.

Targeting structured RNA elements in the SARS-CoV-2 viral genome with small molecules is an attractive strategy for pharmacological control over viral replication. In this work, we report the discovery of small molecules that target the frameshifting element (FSE) in the SARS-CoV-2 RNA genome using high-throughput small-molecule microarray (SMM) screening. A new class of aminoquinazoline ligands for the SARS-CoV-2 FSE are synthesized and characterized using multiple orthogonal biophysical assays and structure-activity relationship (SAR) studies. This work reveals compounds with mid-micromolar binding affinity (KD = 60 ± 6 μM) to the FSE RNA and supports a binding mode distinct from previously reported FSE binders MTDB and merafloxacin. In addition, compounds are active in in vitro dual-luciferase and in-cell dual-fluorescent-reporter frameshifting assays, highlighting the promise of targeting structured elements of RNAs with druglike compounds to alter expression of viral proteins.

Inter- and Intramolecular RNA–RNA Interactions Modulate the Regulation of Translation Mediated by the 3′ UTR in West Nile Virus

RNA viruses rely on genomic structural elements to accomplish the functions necessary to complete the viral cycle. These elements participate in a dynamic network of RNA-RNA interactions that determine the overall folding of the RNA genome and may be responsible for the fine regulation of viral replication and translation as well as the transition between them. The genomes of members of the genus Flavivirus are characterized by a complexly folded 3' UTR with a number of RNA structural elements that are conserved across isolates of each species. The present work provides evidence of intra- and intermolecular RNA-RNA interactions involving RNA structural elements in the 3' UTR of the West Nile virus genome. The intermolecular interactions can be visualized in vitro by the formation of molecular dimers involving the participation of at least the SLI and 3'DB elements. Certainly, the 3' UTR of dengue virus, which lacks the SLI element, forms molecular dimers in lower quantities via a single interaction site, probably 3'DB. The functional analysis of sequence or deletion mutants revealed an inverse relationship between 3' UTR dimerization and viral translation efficiency in cell cultures. A network of RNA-RNA interactions involving 3' UTR structural elements might therefore exist, helping to regulate viral translation.

Aberrant splicing in human cancer: An RNA structural code point of view.

Alternative splicing represents an essential process that occurs widely in eukaryotes. In humans, most genes undergo alternative splicing to ensure transcriptome and proteome diversity reflecting their functional complexity. Over the last decade, aberrantly spliced transcripts due to mutations in cis- or trans-acting splicing regulators have been tightly associated with cancer development, largely drawing scientific attention. Although a plethora of single proteins, ribonucleoproteins, complexed RNAs, and short RNA sequences have emerged as nodal contributors to the splicing cascade, the role of RNA secondary structures in warranting splicing fidelity has been underestimated. Recent studies have leveraged the establishment of novel high-throughput methodologies and bioinformatic tools to shed light on an additional layer of splicing regulation in the context of RNA structural elements. This short review focuses on the most recent available data on splicing mechanism regulation on the basis of RNA secondary structure, emphasizing the importance of the complex RNA G-quadruplex structures (rG4s), and other specific RNA motifs identified as splicing silencers or enhancers. Moreover, it intends to provide knowledge on newly established techniques that allow the identification of RNA structural elements and highlight the potential to develop new RNA-oriented therapeutic strategies against cancer.

Screening for small molecule inhibitors of SAH nucleosidase using an SAH riboswitch

Due to the emergence of multidrug resistant pathogens, it is imperative to identify new targets for antibiotic drug discovery. The S-adenosylhomocysteine (SAH) nucleosidase enzyme is a promising target for antimicrobial drug development due to its critical functions in multiple bacterial processes including recycling of toxic byproducts of S-adenosylmethionine (SAM)-mediated reactions and producing the precursor of the universal quorum sensing signal, autoinducer-2 (AI-2). Riboswitches are structured RNA elements typically used by bacteria to precisely monitor and respond to changes in essential bacterial processes, including metabolism. Natural riboswitches fused to a reporter gene can be exploited to detect changes in metabolism or in physiological signaling. We performed a high-throughput screen (HTS) using an SAH-riboswitch controlled β-galactosidase reporter gene in Escherichia coli to discover small molecules that inhibit SAH recycling. We demonstrate that the assay strategy using SAH riboswitches to detect the effects of SAH nucleosidase inhibitors can quickly identify compounds that penetrate the barriers of Gram-negative bacterial cells and perturb pathways involving SAH.

The recognition of structured elements by a conserved groove distant from domains associated with catalysis is an essential determinant of RNase E.

RNase E is an endoribonuclease found in many bacteria, including important human pathogens. Within Escherichia coli, it has been shown to have a major role in both the maturation of all classes of RNA involved in translation and the initiation of mRNA degradation. Thus, knowledge of the major determinants of RNase E cleavage is central to our understanding and manipulation of bacterial gene expression. We show here that the binding of RNase E to structured RNA elements is crucial for the processing of tRNA, can activate catalysis and may be important in mRNA degradation. The recognition of structured elements by RNase E is mediated by a recently discovered groove that is distant from the domains associated with catalysis. The functioning of this groove is shown here to be essential for E. coli cell viability and may represent a key point of evolutionary divergence from the paralogous RNase G family, which we show lack amino acid residues conserved within the RNA-binding groove of members of the RNase E family. Overall, this work provides new insights into the recognition and cleavage of RNA by RNase E and provides further understanding of the basis of RNase E essentiality in E. coli.

Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seq.

RNA decay serves as a crucial mechanism for maintaining cellular homeostasis and regulating gene expression. Large-scale analyses indicate that altered rates of decay contribute significantly to changes in mRNA levels, with up to half of these changes attributed to decay. The regulation of RNA decay is, at least in part, through structured RNA elements, especially in the non-coding regions of the mRNAs. The development of next-generation sequencing, and in vivo chemical probing techniques has allowed for unprecedented understanding of RNA folding in vivo and genome-wide. To explore the RNA structure elements that are responsible for RNA cleavage, we need to capture the RNA structure before cleavage. In this method, we introduce a new experimental procedure called CAP-STRUCTURE-seq, a modified STRUCTURE-Seq approach combining with the enrichment of in intact mRNAs by the use of terminator exonuclease treatment (5'-Phosphate-Dependent Exonuclease) that digests RNA containing 5-monophosphate ends. This approach is designed to investigate the RNA structure for these intact RNAs, providing a means to study the impact of RNA structure on RNA decay in greater detail. This method can provide insights into the function of RNA structure in RNA decay and help advance our understanding of biological processes.