RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell’s RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5′ end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3′ end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction’s fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production.

Download Full-text

RNA Packing Specificity and Folding during Assembly of the Bacteriophage MS2

Computational and Mathematical Methods in Medicine ◽

10.1080/17486700802168445 ◽

2008 ◽

Vol 9 (3-4) ◽

pp. 339-349 ◽

Cited By ~ 11

Author(s):

Ottar Rolfsson ◽

Katerina Toropova ◽

Victoria Morton ◽

Simona Francese ◽

Gabriella Basnak ◽

...

Keyword(s):

Rna Structure ◽

3D Structure ◽

Viral Assembly ◽

Genomic Rna ◽

Stem Loop ◽

Bacteriophage Ms2 ◽

Protein Dimers ◽

Phage Capsid ◽

Type Phage ◽

Viral Genomic Rna

Using a combination of biochemistry, mass spectrometry, NMR spectroscopy and cryo-electron microscopy (cryo-EM), we have been able to show that quasi-equivalent conformer switching in the coat protein (CP) of an RNA bacteriophage (MS2) is controlled by a sequence-specific RNA–protein interaction. The RNA component of this complex is an RNA stem-loop encompassing just 19 nts from the phage genomic RNA, which is 3569 nts in length. This binding results in the conversion of a CP dimer from a symmetrical conformation to an asymmetric one. Only when both symmetrical and asymmetrical dimers are present in solution is assembly of theT = 3 phage capsid efficient. This implies that the conformers, we have characterized by NMR correspond to the two distinct quasi-equivalent conformers seen in the 3D structure of the virion. An icosahedrally-averaged single particle cryo-EM reconstruction of the wild-type phage (to ∼9 Å resolution) has revealed icosahedrally ordered density encompassing up to 90% of the single-stranded RNA genome. The RNA is seen with a novel arrangement of two concentric shells, with connections between them along the 5-fold symmetry axes. RNA in the outer shell interacts with each of the 90 CP dimers in theT = 3 capsid and although the density is icosahedrally averaged, there appears to be a different average contact at the different quasi-equivalent protein dimers: precisely the result that would be expected if protein conformer switching is RNA-mediated throughout the assembly pathway. This unprecedented RNA structure provides new constraints for models of viral assembly and we describe experiments aimed at probing these. Together, these results suggest that viral genomic RNA folding is an important factor in efficient assembly, and further suggest that RNAs that could sequester viral CPs but not fold appropriately could act as potent inhibitors of viral assembly.

Download Full-text

The global and local distribution of RNA structure throughout the SARS-CoV-2 genome

Journal of Virology ◽

10.1128/jvi.02190-20 ◽

2020 ◽

Cited By ~ 2

Author(s):

Rafael de Cesaris Araujo Tavares ◽

Gandhar Mahadeshwar ◽

Han Wan ◽

Nicholas C. Huston ◽

Anna Marie Pyle

Keyword(s):

In Silico ◽

Rna Structure ◽

Drug Targets ◽

Rna Folding ◽

Rna Viruses ◽

Viral Agent ◽

Rna Structures ◽

Viral Rnas ◽

Viral Genomes ◽

Rna Genome

SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ∼30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to predict regions of high base pair content across long genomes and to pinpoint hotspots of well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity for stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most structured viral RNAs in nature. Furthermore, our analysis suggests varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts, which given the technical difficulty of experimentally separating cellular mixtures of sgRNA from gRNA, is a unique advantage of our in-silico pipeline. The resulting findings provide a useful roadmap for planning focused empirical studies of SARS-CoV-2 RNA biology, and a preliminary guide for exploring potential SARS-CoV-2 RNA drug targets. Importance The RNA genome of SARS-CoV-2 is among the largest and most complex viral genomes, and yet its RNA structural features remain relatively unexplored. Since RNA elements guide function in most RNA viruses, and they represent potential drug targets, it is essential to chart the architectural features of SARS-CoV-2 and pinpoint regions that merit focused study. Here we show that RNA folding stability of SARS-CoV-2 genome is exceptional among viral genomes and we develop a method to directly compare levels of predicted secondary structure across SARS-CoV-2 domains. Remarkably, we find that coding regions display the highest structural propensity in the genome, forming motifs that differ between the genomic and subgenomic contexts. Our approach provides an attractive strategy to rapidly screen for candidate structured regions based on base pairing potential and provides a readily interpretable roadmap to guide functional studies of RNA viruses and other pharmacologically relevant RNA transcripts.

Download Full-text

Cooperativity and Interdependency between RNA Structure and RNA–RNA Interactions

Non-Coding RNA ◽

10.3390/ncrna7040081 ◽

2021 ◽

Vol 7 (4) ◽

pp. 81

Author(s):

Ilias Skeparnias ◽

Jinwei Zhang

Keyword(s):

Rna Structure ◽

Noncoding Rna ◽

Quaternary Structure ◽

Reciprocal Relationship ◽

Rna Structures ◽

Granule Formation ◽

Expression Control ◽

Gene Expression Control ◽

Transcription Complexes ◽

Rna Complexes

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions.

Download Full-text

Cotranscriptional kinetic folding of RNA secondary structures

10.1101/2020.07.10.196972 ◽

2020 ◽

Author(s):

Vo Hong Thanh ◽

Pekka Orponen

Keyword(s):

Structure Formation ◽

Rna Structure ◽

Rna Folding ◽

Computational Prediction ◽

Simulation Method ◽

Rna Structures ◽

Rna Molecules ◽

Primitive Operation ◽

Nucleotide Resolution ◽

Kinetics Of

Computational prediction of RNA structures is an important problem in computational structural biology. Studies of RNA structure formation often assume that the process starts from a fully synthesized sequence. Experimental evidence, however, has shown that RNA folds concurrently with its elongation. We investigate RNA structure formation, taking into account also the cotranscriptional effects. We propose a single-nucleotide resolution kinetic model of the folding process of RNA molecules, where the polymerase-driven elongation of an RNA strand by a new nucleotide is included as a primitive operation, together with a stochastic simulation method that implements this folding concurrently with the transcriptional synthesis. Numerical case studies show that our cotranscriptional RNA folding model can predict the formation of metastable conformations that are favored in actual biological systems. Our new computational tool can thus provide quantitative predictions and offer useful insights into the kinetics of RNA folding.

Download Full-text

Distributed Biotin-Streptavidin Transcription Roadblocks for Mapping Cotranscriptional RNA Folding

10.1101/100073 ◽

2017 ◽

Author(s):

Eric J. Strobel ◽

Kyle E. Watters ◽

Julius B. Lucks

Keyword(s):

Experimental Study ◽

Rna Structure ◽

Rna Folding ◽

Folding Pathway ◽

Decision Making Process ◽

Important Process ◽

Rna Structures ◽

Rna Molecules ◽

Fundamental Properties ◽

Nucleotide Resolution

AbstractRNA molecules fold cotranscriptionally as they emerge from RNA polymerase. Cotranscriptional folding is an important process for proper RNA structure formation as the order of folding can determine an RNA molecule’s structure, and thus its functional properties. Despite its fundamental importance, the experimental study of RNA cotranscriptional folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structures at nucleotide resolution during transcription. We previously developed cotranscriptional selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-seq) to simultaneously probe all of the intermediate structures an RNA molecule transitions through during transcription elongation. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent streptavidin roadblocking strategy to simplify the preparation of roadblocking transcription templates. We determine the fundamental properties of streptavidin roadblocks and show that randomly distributed streptavidin roadblocks can be used in cotranscriptional SHAPE-Seq experiments to measure the Bacillus cereus crcB fluoride riboswitch folding pathway. Comparison of EcoRIE111Q and streptavidin roadblocks in cotranscriptional SHAPE-Seq data shows that both strategies identify the same RNA structural transitions related to the riboswitch decision-making process. Finally, we propose guidelines to leverage the complementary strengths of each transcription roadblock for use in studying cotranscriptional folding.

Download Full-text

Characteristics and Prediction of RNA Structure

BioMed Research International ◽

10.1155/2014/690340 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10

Author(s):

Hengwu Li ◽

Daming Zhu ◽

Caiming Zhang ◽

Huijian Han ◽

Keith A. Crandall

Keyword(s):

Rna Structure ◽

Structure Prediction ◽

Rna Folding ◽

Golden Section ◽

Hierarchical Structures ◽

Secondary Structures ◽

Sequence Length ◽

Rna Structures ◽

Rna Secondary Structures ◽

Hierarchical Folding

RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA STRAND, which are validated by NMR or X-ray. The length ratios of domains in these sequences are approximately 0.382L, 0.5L, 0.618L, andL, whereLis the sequence length. These points are just the important golden sections of sequence. With this characteristic, an algorithm is designed to predict RNA hierarchical structures and simulate RNA folding by dynamically folding RNA structures according to the above golden section points. The sensitivity and number of predicted pseudoknots of our algorithm are better than those of the Mfold, HotKnots, McQfold, ProbKnot, and Lhw-Zhu algorithms. Experimental results reflect the folding rules of RNA from a new angle that is close to natural folding.

Download Full-text

Author response: RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

10.7554/elife.01892.021 ◽

2014 ◽

Author(s):

Erich G Chapman ◽

Stephanie L Moon ◽

Jeffrey Wilusz ◽

Jeffrey S Kieft

Keyword(s):

Dengue Virus ◽

Author Response ◽

Rna Structures ◽

Virus Rna

Download Full-text

Field-deployable molecular diagnostic platform for arbovirus detection in Aedes aegypti

Parasites & Vectors ◽

10.1186/s13071-020-04357-y ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Natalie Rutkowski ◽

Yuemei Dong ◽

George Dimopoulos

Keyword(s):

Aedes Aegypti ◽

Dengue Virus ◽

Real Time ◽

Real Time Pcr ◽

Diagnostic System ◽

Molecular Diagnostic ◽

Infection Prevalence ◽

The Novel ◽

Genomic Rna ◽

Viral Genomic Rna

Abstract Background Surveillance of mosquito infection status is critical for planning and deployment of proper mosquito control initiatives. Point-of-care (POC) detection assays are necessary for monitoring the infection prevalence and geographical range of viruses in mosquito vector populations. We therefore assessed the novel real-time PCR (qPCR) bCUBE (Hyris, London, UK) molecular diagnostic system as a tool for virus detection. Methods Aedes aegypti Rps17 was used to validate and determine correlation coefficient for the novel bCUBE qPCR system to a laboratory standard StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA). Experimentally infected Ae. aegypti were quantified for Zika (ZIKV) and dengue virus serotype 2 (DENV2) viral genomic RNA. Infection prevalence was compared to plaque assay. Results We developed and validated a novel qPCR system for the detection of ZIKV and DENV2 using the real-time qPCR system bCUBE. With bCUBE-based qRT-PCR, viral genomic RNA could be detected in individually infected Ae. aegypti mosquitoes and in pools of 5, 10 or 15 mosquitoes. Conclusions The portable qPCR bCUBE diagnostic system is capable of detecting Zika and dengue virus in mosquitoes and therefore has potential as a practical field-deployable diagnostic test for vector-borne disease surveillance programmes.

Download Full-text

A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation

Nucleic Acids Research ◽

10.1093/nar/gkz668 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9329-9342 ◽

Cited By ~ 5

Author(s):

Chaminda D Gunawardene ◽

Laura R Newburn ◽

K Andrew White

Keyword(s):

Rna Structure ◽

Rna Viruses ◽

Degradation Products ◽

Rna Degradation ◽

Viral Rna ◽

Tobacco Necrosis Virus ◽

Rna Structures ◽

Necrosis Virus

Abstract Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

Download Full-text