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 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 mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis inArabidopsis thaliana

2020 ◽  
Vol 117 (35) ◽  
pp. 21785-21795 ◽  
Author(s):  
Susheel Sagar Bhat ◽  
Dawid Bielewicz ◽  
Tomasz Gulanicz ◽  
Zsuzsanna Bodi ◽  
Xiang Yu ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Structure ◽  
Methylation Status ◽  
Mirna Biogenesis ◽  
Stem Loop ◽  
Structure Probing ◽  
Plant Transcriptome ◽  
Loop Regions

InArabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introducesN6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem–loop regions of these transcripts inmtamutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA inmtamutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes ofmtamutant plants.

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 Translation of the Flavivirus Kunjin NS3 Gene in cis but Not Its RNA Sequence or Secondary Structure Is Essential for Efficient RNA Packaging

2006 ◽  
Vol 80 (22) ◽  
pp. 11255-11264 ◽  
Author(s):  
Gorben P. Pijlman ◽  
Natasha Kondratieva ◽  
Alexander A. Khromykh
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Rna Replication ◽  
Full Length ◽  
Rna Structures ◽  
Coding Region ◽  
Rna Packaging ◽  
Virus Like Particles ◽  
Virus Particles ◽  
Ns3 Protein

ABSTRACT Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

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 RNA secondary structure prediction using an ensemble of two-dimensional deep neural networks and transfer learning

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jaswinder Singh ◽  
Jack Hanson ◽  
Kuldip Paliwal ◽  
Yaoqi Zhou
Keyword(s):  
Secondary Structure ◽  
Transfer Learning ◽  
Rna Structure ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Noncoding Rnas ◽  
Rna Structures ◽  
Base Pairs ◽  
Functional Annotations ◽  
Model Training

AbstractThe majority of our human genome transcribes into noncoding RNAs with unknown structures and functions. Obtaining functional clues for noncoding RNAs requires accurate base-pairing or secondary-structure prediction. However, the performance of such predictions by current folding-based algorithms has been stagnated for more than a decade. Here, we propose the use of deep contextual learning for base-pair prediction including those noncanonical and non-nested (pseudoknot) base pairs stabilized by tertiary interactions. Since only $$<$$<250 nonredundant, high-resolution RNA structures are available for model training, we utilize transfer learning from a model initially trained with a recent high-quality bpRNA dataset of $$> $$>10,000 nonredundant RNAs made available through comparative analysis. The resulting method achieves large, statistically significant improvement in predicting all base pairs, noncanonical and non-nested base pairs in particular. The proposed method (SPOT-RNA), with a freely available server and standalone software, should be useful for improving RNA structure modeling, sequence alignment, and functional annotations.

An efficient algorithm for planar drawing of RNA structures with pseudoknots of any type

2016 ◽  
Vol 14 (03) ◽  
pp. 1650009 ◽  
Author(s):  
Yanga Byun ◽  
Kyungsook Han
Keyword(s):  
Rna Structure ◽  
Large Scale ◽  
Structural Elements ◽  
Rna Structures ◽  
Structural Element ◽  
Rna Pseudoknots ◽  
Number Of Stems ◽  
Rna Pseudoknot ◽  
Aesthetic Structure ◽  
Planar Drawing

An RNA pseudoknot is a tertiary structural element in which bases of a loop pair with complementary bases are outside the loop. A drawing of RNA secondary structures is a tree, but a drawing of RNA pseudoknots is a graph that has an inner cycle within a pseudoknot and possibly outer cycles formed between the pseudoknot and other structural elements. Visualizing a large-scale RNA structure with pseudoknots as a planar drawing is challenging because a planar drawing of an RNA structure requires both pseudoknots and an entire structure enclosing the pseudoknots to be embedded into a plane without overlapping or crossing. This paper presents an efficient heuristic algorithm for visualizing a pseudoknotted RNA structure as a planar drawing. The algorithm consists of several parts for finding crossing stems and page mapping the stems, for the layout of stem-loops and pseudoknots, and for overlap detection between structural elements and resolving it. Unlike previous algorithms, our algorithm generates a planar drawing for a large RNA structure with pseudoknots of any type and provides a bracket view of the structure. It generates a compact and aesthetic structure graph for a large pseudoknotted RNA structure in O([Formula: see text]) time, where n is the number of stems of the RNA structure.

scholarly journals Current perspectives on RNA secondary structure probing

2014 ◽  
Vol 42 (4) ◽  
pp. 1251-1255 ◽  
Author(s):  
Julia Kenyon ◽  
Liam Prestwood ◽  
Andrew Lever
Keyword(s):  
Structural Analysis ◽  
Secondary Structure ◽  
Rna Structure ◽  
Rna Secondary Structure ◽  
Biological Systems ◽  
Present Review ◽  
Structure Probing ◽  
Recent Advances ◽  
Near Future

The range of roles played by structured RNAs in biological systems is vast. At the same time as we are learning more about the importance of RNA structure, recent advances in reagents, methods and technology mean that RNA secondary structural probing has become faster and more accurate. As a result, the capabilities of laboratories that already perform this type of structural analysis have increased greatly, and it has also become more widely accessible. The present review summarizes established and recently developed techniques. The information we can derive from secondary structural analysis is assessed, together with the areas in which we are likely to see exciting developments in the near future.

scholarly journals FoldAtlas: a repository for genome-wide RNA structure probing data

2016 ◽  
Vol 33 (2) ◽  
pp. 306-308 ◽  
Author(s):  
Matthew Norris ◽  
Chun Kit Kwok ◽  
Jitender Cheema ◽  
Matthew Hartley ◽  
Richard J. Morris ◽  
...  
Keyword(s):  
Rna Structure ◽  
Structure Probing ◽  
Genome Wide

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.

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