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.

scholarly journals Comparative analysis of coronavirus genomic RNA structure reveals conservation in SARS-like coronaviruses

2020 ◽  
Author(s):  
Wes Sanders ◽  
Ethan J. Fritch ◽  
Emily A. Madden ◽  
Rachel L. Graham ◽  
Heather A. Vincent ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Structure ◽  
Rna Secondary Structure ◽  
Molecular Mechanisms ◽  
Selective Pressure ◽  
Secondary Structures ◽  
Rna Structures ◽  
Rna Secondary Structures ◽  
The Past ◽  
Novel Strategy

AbstractCoronaviruses, including SARS-CoV-2 the etiological agent of COVID-19 disease, have caused multiple epidemic and pandemic outbreaks in the past 20 years1–3. With no vaccines, and only recently developed antiviral therapeutics, we are ill equipped to handle coronavirus outbreaks4. A better understanding of the molecular mechanisms that regulate coronavirus replication and pathogenesis is needed to guide the development of new antiviral therapeutics and vaccines. RNA secondary structures play critical roles in multiple aspects of coronavirus replication, but the extent and conservation of RNA secondary structure across coronavirus genomes is unknown5. Here, we define highly structured RNA regions throughout the MERS-CoV, SARS-CoV, and SARS-CoV-2 genomes. We find that highly stable RNA structures are pervasive throughout coronavirus genomes, and are conserved between the SARS-like CoV. Our data suggests that selective pressure helps preserve RNA secondary structure in coronavirus genomes, suggesting that these structures may play important roles in virus replication and pathogenesis. Thus, disruption of conserved RNA secondary structures could be a novel strategy for the generation of attenuated SARS-CoV-2 vaccines for use against the current COVID-19 pandemic.

scholarly journals Using SHAPE-MaP To Model RNA Secondary Structure and Identify 3′UTR Variation in Chikungunya Virus

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Emily A. Madden ◽  
Kenneth S. Plante ◽  
Clayton R. Morrison ◽  
Katrina M. Kutchko ◽  
Wes Sanders ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Virus Replication ◽  
Rna Structure ◽  
Rna Secondary Structure ◽  
Chikungunya Virus ◽  
Secondary Structures ◽  
Rna Structures ◽  
Functional Importance ◽  
Rna Secondary Structures ◽  
Mosquito Cells

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3′UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3′UTR variants. A duplication in the 3′UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3′UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3′UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.

scholarly journals Amount of RNA secondary structure required to induce an alternative splice.

1987 ◽  
Vol 7 (9) ◽  
pp. 3194-3198 ◽  
Author(s):  
D Solnick ◽  
S I Lee
Keyword(s):  
Alternative Splicing ◽  
Secondary Structure ◽  
Alternative Splice ◽  
Rna Secondary Structure ◽  
Secondary Structures ◽  
Splice Sites ◽  
Perfect Complementarity ◽  
Set Up

We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than approximately 50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.

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.

Amount of RNA secondary structure required to induce an alternative splice

1987 ◽  
Vol 7 (9) ◽  
pp. 3194-3198
Author(s):  
D Solnick ◽  
S I Lee
Keyword(s):  
Alternative Splicing ◽  
Secondary Structure ◽  
Alternative Splice ◽  
Rna Secondary Structure ◽  
Secondary Structures ◽  
Splice Sites ◽  
Perfect Complementarity ◽  
Set Up

We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than approximately 50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.

scholarly journals RNA secondary structure prediction using deep learning with thermodynamic integration

2020 ◽  
Author(s):  
Kengo Sato ◽  
Manato Akiyama ◽  
Yasubumi Sakakibara
Keyword(s):  
Deep Learning ◽  
Secondary Structure ◽  
Structure Prediction ◽  
Rna Secondary Structure ◽  
Secondary Structure Prediction ◽  
Secondary Structures ◽  
Thermodynamic Integration ◽  
Rna Secondary Structure Prediction ◽  
Rna Secondary Structures ◽  
Non Coding Rnas

RNA secondary structure prediction is one of the key technologies for revealing the essential roles of functional non-coding RNAs. Although machine learning-based rich-parametrized models have achieved extremely high performance in terms of prediction accuracy, the risk of overfitting for such models has been reported. In this work, we propose a new algorithm for predicting RNA secondary structures that uses deep learning with thermodynamic integration, thereby enabling robust predictions. Similar to our previous work, the folding scores, which are computed by a deep neural network, are integrated with traditional thermodynamic parameters to enable robust predictions. We also propose thermodynamic regularization for training our model without overfitting it to the training data. Our algorithm (MXfold2) achieved the most robust and accurate predictions in computational experiments designed for newly discovered non-coding RNAs, with significant 2–10 % improvements over our previous algorithm (MXfold) and standard algorithms for predicting RNA secondary structures in terms of F-value.

scholarly journals RNA Secondary Structurome Revealed Distinct Thermoregulation in Plasmodium falciparum

2022 ◽  
Vol 9 ◽  
Author(s):  
Yanwei Qi ◽  
Yuhong Zhang ◽  
Quankai Mu ◽  
Guixing Zheng ◽  
Mengxin Zhang ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Secondary Structure ◽  
Rna Structure ◽  
Rna Secondary Structure ◽  
Structural Changes ◽  
Environmental Changes ◽  
Parasite Development ◽  
Temperature Dependent ◽  
Trophozoite Stage ◽  
Temperature Changes

The development of Plasmodium parasites, a causative agent of malaria, requests two hosts and the completion of 11 different parasite stages during development. Therefore, an efficient and fast response of parasites to various complex environmental changes, such as ambient temperature, pH, ions, and nutrients, is essential for parasite development and survival. Among many of these environmental changes, temperature is a decisive factor for parasite development and pathogenesis, including the thermoregulation of rRNA expression, gametogenesis, and parasite sequestration in cerebral malaria. However, the exact mechanism of how Plasmodium parasites rapidly respond and adapt to temperature change remains elusive. As a fundamental and pervasive regulator of gene expression, RNA structure can be a specific mechanism for fine tuning various biological processes. For example, dynamic and temperature-dependent changes in RNA secondary structures can control the expression of different gene programs, as shown by RNA thermometers. In this study, we applied the in vitro and in vivo transcriptomic-wide secondary structurome approach icSHAPE to measure parasite RNA structure changes with temperature alteration at single-nucleotide resolution for ring and trophozoite stage parasites. Among 3,000 probed structures at different temperatures, our data showed structural changes in the global transcriptome, such as S-type rRNA, HRPII gene, and the erythrocyte membrane protein family. When the temperature drops from 37°C to 26°C, most of the genes in the trophozoite stage cause significantly more changes to the RNA structure than the genes in the ring stage. A multi-omics analysis of transcriptome data from RNA-seq and RNA structure data from icSHAPE reveals that the specific RNA secondary structure plays a significant role in the regulation of transcript expression for parasites in response to temperature changes. In addition, we identified several RNA thermometers (RNATs) that responded quickly to temperature changes. The possible thermo-responsive RNAs in Plasmodium falciparum were further mapped. To this end, we identified dynamic and temperature-dependent RNA structural changes in the P. falciparum transcriptome and performed a comprehensive characterization of RNA secondary structures over the course of temperature stress in blood stage development. These findings not only contribute to a better understanding of the function of the RNA secondary structure but may also provide novel targets for efficient vaccines or drugs.

Visualization of RNA secondary structure with pseudoknots

2021 ◽  
pp. 2150036
Author(s):  
Lina Yang ◽  
Yang Liu ◽  
Huiwu Luo ◽  
Xichun Li ◽  
Yuan Yan Tang
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Large Scale ◽  
Secondary Structures ◽  
Combination Method ◽  
Joint Analysis ◽  
Discrete Wavelet ◽  
Visualization Method ◽  
Number Representation ◽  
Rna Secondary Structures

The function of pseudoknots cannot be ignored in the RNA secondary structure. Existing methods for analyzing RNA secondary structures with pseudoknots exhibit many shortcomings. This paper presents a novel RNA secondary structure visualization method in the case of a joint analysis of RNA primary structures and secondary structures. The way is based on the page number representation of the RNA secondary structure. It innovatively uses five vectors to represent bases, which are sequentially connected to outline the characteristics of the RNA secondary structure. The method covers almost all the constituent elements of the RNA secondary structure and extracts features completely. Experiments are based on the available techniques for large-scale annotation of RNA secondary structures, using a combination method of discrete wavelet transform and fractal dimension. The classification effect is compared with the previous RNA secondary structure representation methods. Experimental results show that the RNA secondary structure visualization method proposed in this paper has good application prospects in RNA secondary structure classification.

RNA In Silico The Computational Biology of RNA Secondary Structures

1999 ◽  
Vol 02 (01) ◽  
pp. 65-90 ◽  
Author(s):  
Chirstoph Flamm ◽  
Ivo L. Hofacker ◽  
Peter F. Stadler
Keyword(s):  
Secondary Structure ◽  
Fitness Landscape ◽  
Rna Virus ◽  
Point Mutations ◽  
Secondary Structures ◽  
Hill Climbing ◽  
Stable States ◽  
Punctuated Equilibria ◽  
Rna Secondary Structures

RNA secondary structures provide a unique computer model for investigating the most important aspects of structural and evolutionary biology. The existence of efficient algorithms for solving the folding problem, i.e., for predicting the secondary structure given only the sequence, allows the construction of realistic computer simulations. The notion of a "landscape" underlies both the structure formation (folding) and the (in vitro) evolution of RNA. Evolutionary adaptation may be seen as hill climbing process on a fitness landscape which is determined by the phenotype of the RNA molecule (within the model this is its secondary structure) and the selection constraints acting on the molecules. We find that a substantial fraction of point mutations do not change an RNA secondary structure. On the other hand, a comparable fraction of mutations leads to very different structures. This interplay of smoothness and ruggedness (or robustness and sensitivity) is a generic feature of both RNA and protein sequence-structure maps. Its consequences, "shape space covering" and "neutral networks" are inherited by the fitness landscapes and determine the dynamics of RNA evolution. Punctuated equilibria at phenotype level and a diffusion like evolution of the underlying genotypes are a characteristics feature of such models. As a practical application of these theoretical findings we have designed an algorithm that finds conserved (and therefore potentially functional substructures of RNA virus genomes from spares data sets. The folding dynamics of particular RNA molecule can also be studied successfully based on secondary structure. Given an RNA sequence, we consider the energy landscape formed by all possible conformations (secondary structures). A straight formward implementation of the Metropolis algorithm is sufficient to produce a quite realistic folding kinetics, allowing to identify meta-stable states and folding pathways. Just as in the protein case there are good and bad folders which can be distinguished by the properties of their landscapes.

scholarly journals A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

2021 ◽  
Author(s):  
Tycho Marinus ◽  
Adam B Fessler ◽  
Craig A Ogle ◽  
Danny Incarnato
Keyword(s):  
Rna Structure ◽  
Structure Prediction ◽  
Critical Role ◽  
Living Cells ◽  
Pathological Process ◽  
Rna Structures ◽  
Rna Molecules ◽  
Derived Data

Abstract Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

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