scholarly journals Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

2020 ◽  
Author(s):  
Nicholas C. Huston ◽  
Han Wan ◽  
Rafael de Cesaris Araujo Tavares ◽  
Craig Wilen ◽  
Anna Marie Pyle
Keyword(s):  
Secondary Structure ◽  
Rna Virus ◽  
Genomic Structure ◽  
Structural Features ◽  
Evolutionary Analysis ◽  
Rna Structures ◽  
Genomic Context ◽  
Positive Sense ◽  
Folded Rna

SummarySARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.

scholarly journals Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

2004 ◽  
Vol 78 (14) ◽  
pp. 7619-7633 ◽  
Author(s):  
Guohua Zhang ◽  
Jiuchun Zhang ◽  
Anne E. Simon
Keyword(s):  
Solution Structure ◽  
Rna Virus ◽  
Structural Features ◽  
General Mechanism ◽  
Minus Strand ◽  
Turnip Crinkle Virus ◽  
Structural Elements ◽  
Transcription In Vitro

ABSTRACT Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3′-terminal bases. Deletion of satC 3′-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3′ end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3′-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3′ terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3′ end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3′-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus.

scholarly journals Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

2017 ◽  
Vol 46 (5) ◽  
pp. 2573-2584 ◽  
Author(s):  
Kyle E Watters ◽  
Krishna Choudhary ◽  
Sharon Aviran ◽  
Julius B Lucks ◽  
Keith L Perry ◽  
...  
Keyword(s):  
Rna Virus ◽  
Structural Elements ◽  
Rna Structures ◽  
Selection Pressures ◽  
Positive Sense

scholarly journals Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements

2020 ◽  
Vol 48 (22) ◽  
pp. 12436-12452 ◽  
Author(s):  
Ilaria Manfredonia ◽  
Chandran Nithin ◽  
Almudena Ponce-Salvatierra ◽  
Pritha Ghosh ◽  
Tomasz K Wirecki ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Therapeutic Strategies ◽  
Rna Structures ◽  
Genome Wide ◽  
High Sequence Conservation ◽  
Infected Cells ◽  
Rna Elements

Abstract SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5′ UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

scholarly journals Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

2000 ◽  
Vol 74 (10) ◽  
pp. 4590-4600 ◽  
Author(s):  
Ian Goodfellow ◽  
Yasmin Chaudhry ◽  
Andrew Richardson ◽  
Janet Meredith ◽  
Jeffrey W. Almond ◽  
...  
Keyword(s):  
Rna Virus ◽  
Site Directed Mutagenesis ◽  
Evolutionary Significance ◽  
Rna Structures ◽  
Coding Region ◽  
Rna Sequences ◽  
Stem Loop ◽  
Cis Acting ◽  
Function Recovery ◽  
Positive Sense

ABSTRACT The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel polioviruscis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

scholarly journals Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Minjie Zhang ◽  
Kongpan Li ◽  
Jianhui Bai ◽  
Willem A. Velema ◽  
Chengqing Yu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rna Virus ◽  
Genome Structure ◽  
Direct Determination ◽  
Rna Structures ◽  
Disease States ◽  
Ribosome Binding Sites ◽  
Enterovirus D68

AbstractDirect determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (−) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.

scholarly journals Protein Profiles: Biases and Protocols

2020 ◽  
Author(s):  
Gregor Urban ◽  
Mirko Torrisi ◽  
Christophe N. Magnan ◽  
Gianluca Pollastri ◽  
Pierre Baldi
Keyword(s):  
Secondary Structure ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Protein Secondary Structure ◽  
Structural Features ◽  
Profile Similarity ◽  
Redundancy Reduction ◽  
Test Sets

AbstractThe use of evolutionary profiles to predict protein secondary structure, as well as other protein structural features, has been standard practice since the 1990s. Using profiles in the input of such predictors, in place or in addition to the sequence itself, leads to significantly more accurate predictors. While profiles can enhance structural signals, their role remains somewhat surprising as proteins do not use profiles when folding in vivo. Furthermore, the same sequence-based redundancy reduction protocols initially derived to train and evaluate sequence-based predictors, have been applied to train and evaluate profile-based predictors. This can lead to unfair comparisons since profile may facilitate the bleeding of information between training and test sets. Here we use the extensively studied problem of secondary structure prediction to better evaluate the role of profiles and show that: (1) high levels of profile similarity between training and test proteins are observed when using standard sequence-based redundancy protocols; (2) the gain in accuracy for profile-based predictors, over sequence-based predictors, strongly relies on these high levels of profile similarity between training and test proteins; and (3) the overall accuracy of a profile-based predictor on a given protein dataset provides a biased measure when trying to estimate the actual accuracy of the predictor, or when comparing it to other predictors. We show, however, that this bias can be avoided by implementing a new protocol (EVALpro) which evaluates the accuracy of profile-based predictors as a function of the profile similarity between training and test proteins. Such a protocol not only allows for a fair comparison of the predictors on equally hard or easy examples, but also completely removes the need for selecting arbitrary similarity cutoffs when selecting test proteins. The EVALpro program is available for download from the SCRATCH suite (http://scratch.proteomics.ics.uci.edu).

scholarly journals Repurposing Ribo-Seq to provide insights into structured RNAs

2020 ◽  
Author(s):  
Brayon J. Fremin ◽  
Ami S. Bhatt
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Large Scale ◽  
Structural Information ◽  
Ribosome Profiling ◽  
Rna Structures ◽  
Size Selection ◽  
Bacterial Ribosome ◽  
Powerful Approach

AbstractRibosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, this method can enrich RNAs that are not bound by ribosomes, but rather, are protected from degradation in another way. For example, Escherichia coli Ribo-Seq libraries also capture reads from most non-coding RNAs (ncRNAs). These fragments of ncRNAs pass all size selection steps of the Ribo-Seq protocol and survive hours of MNase treatment, presumably without protection from the ribosome or other macromolecules or proteins. Since bacterial ribosome profiling does not directly isolate ribosomes, but instead uses broad size range cutoffs to fractionate actively translated RNAs, it is understandable that some ncRNAs are retained after size selection. However, how these ‘contaminants’ survive MNase treatment is unclear. Through analyzing metaRibo-Seq reads across ssrS, a well established structured RNA in E. coli, and structured direct repeats from Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays in Ruminococcus lactaris, we observed that these RNAs are protected from MNase treatment by virtue of their secondary structure. Therefore, large volumes of data previously discarded as contaminants in bacterial Ribo-Seq experiments can, in fact, be used to gain information regarding the in vivo secondary structure of ncRNAs, providing unique insight into their native functional structures.ImportanceWe observe that ‘contaminant’ signals in bacterial Ribo-Seq experiments that are often disregarded and discarded, in fact, strongly overlap with structured regions of ncRNAs. Structured ncRNAs are pivotal mediators of bioregulation in bacteria and their functions are often reliant on their specific structures. We present an approach to access important RNA structural information through merely repurposing ‘contaminant’ signals in bacterial Ribo-Seq experiments. This powerful approach enables us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large-scale and in vivo.

scholarly journals Genome-wide mapping of therapeutically-relevant SARS-CoV-2 RNA structures

2020 ◽  
Author(s):  
Ilaria Manfredonia ◽  
Chandran Nithin ◽  
Almudena Ponce-Salvatierra ◽  
Pritha Ghosh ◽  
Tomasz K. Wirecki ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Therapeutic Strategies ◽  
Structural Elements ◽  
Rna Structures ◽  
Structure Probing ◽  
Genome Wide ◽  
Positive Sense ◽  
High Sequence Conservation ◽  
Single Base Resolution

SummarySARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome of ∼30 kb, whose outbreak caused the still ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally conserved structural elements within coronavirus RNA genomes have been identified to date.Here, we performed RNA structure probing by SHAPE-MaP to obtain a single-base resolution secondary structure map of the full SARS-CoV-2 coronavirus genome. The SHAPE-MaP probing data recapitulate the previously described coronavirus RNA elements (5′ UTR, ribosomal frameshifting element, and 3′ UTR), and reveal new structures. Secondary structure-restrained 3D modeling of highly-structured regions across the SARS-CoV-2 genome allowed for the identification of several putative druggable pockets. Furthermore, ∼8% of the identified structure elements show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. In addition, we identify a set of persistently single-stranded regions having high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics.Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

scholarly journals Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 525
Author(s):  
Paula Michalak ◽  
Julita Piasecka ◽  
Barbara Szutkowska ◽  
Ryszard Kierzek ◽  
Ewa Biala ◽  
...  
Keyword(s):  
Experimental Data ◽  
Secondary Structure ◽  
Influenza A Virus ◽  
Influenza A ◽  
Chemical Mapping ◽  
Rna Structures ◽  
Structural Motifs ◽  
2009 H1n1

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

scholarly journals In Vivo and In Vitro Genome-Wide Profiling of RNA Secondary Structures Reveals Key Regulatory Features in Plasmodium falciparum

2021 ◽  
Vol 11 ◽  
Author(s):  
Yanwei Qi ◽  
Yuhong Zhang ◽  
Guixing Zheng ◽  
Bingxia Chen ◽  
Mengxin Zhang ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Rna Secondary Structure ◽  
Secondary Structures ◽  
Rna Structures ◽  
Trophozoite Stage ◽  
Rna Secondary Structures ◽  
Biological Programme

It is widely accepted that the structure of RNA plays important roles in a number of biological processes, such as polyadenylation, splicing, and catalytic functions. Dynamic changes in RNA structure are able to regulate the gene expression programme and can be used as a highly specific and subtle mechanism for governing cellular processes. However, the nature of most RNA secondary structures in Plasmodium falciparum has not been determined. To investigate the genome-wide RNA secondary structural features at single-nucleotide resolution in P. falciparum, we applied a novel high-throughput method utilizing the chemical modification of RNA structures to characterize these structures. Structural data from parasites are in close agreement with the known 18S ribosomal RNA secondary structures of P. falciparum and can help to predict the in vivo RNA secondary structure of a total of 3,396 transcripts in the ring-stage and trophozoite-stage developmental cycles. By parallel analysis of RNA structures in vivo and in vitro during the Plasmodium parasite ring-stage and trophozoite-stage intraerythrocytic developmental cycles, we identified some key regulatory features. Recent studies have established that the RNA structure is a ubiquitous and fundamental regulator of gene expression. Our study indicate that there is a critical connection between RNA secondary structure and mRNA abundance during the complex biological programme of P. falciparum. This work presents a useful framework and important results, which may facilitate further research investigating the interactions between RNA secondary structure and the complex biological programme in P. falciparum. The RNA secondary structure characterized in this study has potential applications and important implications regarding the identification of RNA structural elements, which are important for parasite infection and elucidating host-parasite interactions and parasites in the environment.

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