Conserved RNA Secondary Structures Research Articles

Comprehensive survey of conserved RNA secondary structures in full-genome alignment of Hepatitis C virus

Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large ‘cloud’ of RNA genomes (quasispecies) which—by trial and error—comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements. Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies. Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches of Hepacivirus C are provided in the supplement.

E-RNA: a collection of web-servers for the prediction and visualisation of RNA secondary structure and their functional features.

e-RNA is a collection of web-servers for the prediction and visualisation of RNA secondary structures and their functional features, including in particular RNA-RNA interactions. In this updated version, we have added novel tools for RNA secondary structure prediction and have significantly updated the visualisation functionality. The new method CoBold can identify transient RNA structure features and their potential functional effects on a known RNA structure during co-transcriptional structure formation. New tool ShapeSorter can predict evolutionarily conserved RNA secondary structure features while simultaneously taking experimental SHAPE probing evidence into account. The web-server R-Chie which visualises RNA secondary structure information in terms of arc diagrams, can now be used to also visualise and intuitively compare RNA-RNA, RNA-DNA and DNA-DNA interactions alongside multiple sequence alignments and quantitative information. The prediction generated by any method in e-RNA can be readily visualised on the web-server. For completed tasks, users can download their results and readily visualise them later on with R-Chie without having to re-run the predictions. e-RNA can be found at http://www.e-rna.org.

Target Landscape of Conserved Plant MicroRNAs and the Complexities of Their Ancient MicroRNA-Binding Sites.

In plants, microRNA (miRNA)-target interactions (MTIs) require high complementarity, a feature from which bioinformatic programs have predicted numerous and diverse targets for any given miRNA, promoting the idea of complex miRNA networks. Opposing this is a hypothesis of constrained miRNA specificity, in which functional MTIs are restricted to the few targets whose required expression output is compatible with the expression of the miRNA. To explore these opposing views, the bioinformatic pipeline Targets Ranked Using Experimental Evidence was applied to strongly conserved miRNAs to identity their high-evidence (HE) targets across species. For each miRNA family, HE targets predominantly consisted of homologs from one conserved target gene family (primary family). These primary families corresponded to the known canonical miRNA-target families, validating the approach. Very few additional HE target families were identified (secondary family), and if so, they were likely functionally related to the primary family. Many primary target families contained highly conserved nucleotide sequences flanking their miRNA-binding sites that were enriched in HE homologs across species. A number of these flanking sequences are predicted to form conserved RNA secondary structures that preferentially base pair with the miRNA-binding site, implying that these sites are highly structured. Our findings support a target landscape view that is dominated by the conserved primary target families, with a minority of either secondary target families or non-conserved targets. This is consistent with the constrained hypothesis of functional miRNA specificity, which potentially in part is being facilitated by features beyond complementarity.

Conserved RNA secondary structure in Cherry virus A 5′-UTR associated with translation regulation

BackgroundA variety of cis-acting RNA elements with structures in the 5′- or 3′-untranslated region (UTR) of viral genomes play key roles in viral translation. Cherry virus A (CVA) is a member of the genus Capillovirus in the family Betaflexiviridae. It has a positive single-stranded RNA genome of ~ 7400 nucleotides (nt). The length of the CVA 5′-UTR is ~ 100 nt; however, the function of this long UTR has not yet been reported.MethodsMolecular and phylogenetic analyses were performed on 75 CVA sequences, which could be divided into four groups, and the RNA secondary structure was predicted in four CVA 5′-UTR types. These four CVA 5′-UTR types were then inserted upstream of the firefly luciferase reporter gene FLuc (FLuc), and in vitro translation of the corresponding transcripts was evaluated using wheat germ extract (WGE). Then, in-line structure probing was performed to reveal the conserved RNA structures in CVA-5′UTR.ResultsThe four CVA 5′-UTR types appeared to have a conserved RNA structure, and the FLuc construct containing these four CVA 5′-UTR types increased the translation of FLuc by 2–3 folds, suggesting weak translation enhancement activity. Mutations in CVA 5′-UTR suppressed translation, suggesting that the conserved RNA structure was important for function.ConclusionThe conserved RNA secondary structure was identified by structural evolution analysis of different CVA isolates and was found to regulate translation.

Evaluation of Conserved RNA Secondary Structures within and between Geographic Lineages of Zika Virus.

Zika virus (ZIKV), without a vaccine or an effective treatment approved to date, has globally spread in the last century. The infection caused by ZIKV in humans has changed progressively from mild to subclinical in recent years, causing epidemics with greater infectivity, tropism towards new tissues and other related symptoms as a product of various emergent ZIKV–host cell interactions. However, it is still unknown why or how the RNA genome structure impacts those interactions in differential evolutionary origin strains. Moreover, the genomic comparison of ZIKV strains from the sequence-based phylogenetic analysis is well known, but differences from RNA structure comparisons have barely been studied. Thus, in order to understand the RNA genome variability of lineages of various geographic distributions better, 410 complete genomes in a phylogenomic scanning were used to study the conservation of structured RNAs. Our results show the contemporary landscape of conserved structured regions with unique conserved structured regions in clades or in lineages within circulating ZIKV strains. We propose these structures as candidates for further experimental validation to establish their potential role in vital functions of the viral cycle of ZIKV and their possible associations with the singularities of different outbreaks that lead to ZIKV populations to acquire nucleotide substitutions, which is evidence of the local structure genome differentiation.

Classifying Conserved RNA Secondary Structures With Pseudoknots by Vector-Edit Distance

Secondary structures have been proved to relate with the great functional diversity of RNA. There have been many studies to predict and compare the RNA secondary structures. However, fast and accurate comparison of RNA secondary structures with arbitrary pseudoknots is a challenging issue due to the hidden but important structural properties, such as the distribution of stems and branches. In this paper, we construct a novel RNA secondary structure model called modified adjoining grammars binary tree ( $\mathrm {BTMG_{CSP}}$ ). It can not only represent the complex RNA secondary structure including arbitrary pseudoknots intuitively, but also reserve RNA secondary structure properties. Further, we propose vector-edit distance to measure the structure similarity between $\mathrm {BTMG_{CSP}}$ trees converted from RNA sequences and their secondary structures for classifying conserved stem pattern. The experimental results show that our method substantially reduces the memory and time consumption in contrast to previous algorithms, such as $O((n/k)^{2}) $ and $O(n/k) $ for time and space complexity, respectively. In particular, the AUC value of our method achieves 0.949 in PseudoBase.

A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids

Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.

A survey of RNA secondary structural propensity encoded within human herpesvirus genomes: global comparisons and local motifs.

There are nine herpesviruses known to infect humans, of which Epstein–Barr virus (EBV) is the most widely distributed (>90% of adults infected). This ubiquitous virus is implicated in a variety of cancers and autoimmune diseases. Previous analyses of the EBV genome revealed numerous regions with evidence of generating unusually stable and conserved RNA secondary structures and led to the discovery of a novel class of EBV non-coding (nc)RNAs: the stable intronic sequence (sis)RNAs. To gain a better understanding of the roles of RNA structure in EBV biology and pathogenicity, we revisit EBV using recently developed tools for genome-wide motif discovery and RNA structural characterization. This corroborated previous results and revealed novel motifs with potential functionality; one of which has been experimentally validated. Additionally, since many herpesviruses increasingly rival the seroprevalence of EBV (VZV, HHV-6 and HHV-7 being the most notable), analyses were expanded to include all sequenced human Herpesvirus RefSeq genomes, allowing for genomic comparisons. In total 10 genomes were analyzed, for EBV (types 1 and 2), HCMV, HHV-6A, HHV-6B, HHV-7, HSV-1, HSV-2, KSHV, and VZV. All resulting data were archived in the RNAStructuromeDB (https://structurome.bb.iastate.edu/herpesvirus) to make them available to a wide array of researchers.

Impact of virus subtype and host IFNL4 genotype on large-scale RNA structure formation in the genome of hepatitis C virus.

Mechanisms underlying the ability of hepatitis C virus (HCV) to establish persistent infections and induce progressive liver disease remain poorly understood. HCV is one of several positive-stranded RNA viruses capable of establishing persistence in their immunocompetent vertebrate hosts, an attribute previously associated with formation of large-scale RNA structure in their genomic RNA. We developed novel methods to analyze and visualize genome-scale ordered RNA structure (GORS) predicted from the increasingly large data sets of complete genome sequences of HCV. Structurally conserved RNA secondary structure in coding regions of HCV localized exclusively to polyprotein ends (core, NS5B). Coding regions elsewhere were also intensely structured based on elevated minimum folding energy difference (MFED) values, but the actual stem–loop elements involved in genome folding were structurally poorly conserved, even between subtypes 1a and 1b. Dynamic remodeling was further evident from comparison of HCV strains in different host genetic backgrounds. Significantly higher MFED values, greater suppression of UpA dinucleotide frequencies, and restricted diversification were found in subjects with the TT genotype of the rs12979860 SNP in the IFNL4 gene compared to the CC (nonexpressing) allele. These structural and compositional associations with expression of interferon-λ4 were recapitulated on a larger scale by higher MFED values and greater UpA suppression of genotype 1 compared to genotype 3a, associated with previously reported HCV genotype-associated differences in hepatic interferon-stimulated gene induction. Associations between innate cellular responses with HCV structure and further evolutionary constraints represent an important new element in RNA virus evolution and the adaptive interplay between virus and host.

Discovery of 20 novel ribosomal leader candidates in bacteria and archaea

BackgroundRNAs perform many functions in addition to supplying coding templates, such as binding proteins. RNA-protein interactions are important in multiple processes in all domains of life, and the discovery of additional protein-binding RNAs expands the scope for studying such interactions. To find such RNAs, we exploited a form of ribosomal regulation. Ribosome biosynthesis must be tightly regulated to ensure that concentrations of rRNAs and ribosomal proteins (r-proteins) match. One regulatory mechanism is a ribosomal leader (r-leader), which is a domain in the 5′ UTR of an mRNA whose genes encode r-proteins. When the concentration of one of these r-proteins is high, the protein binds the r-leader in its own mRNA, reducing gene expression and thus protein concentrations. To date, 35 types of r-leaders have been validated or predicted.ResultsBy analyzing additional conserved RNA structures on a multi-genome scale, we identified 20 novel r-leader structures. Surprisingly, these included new r-leaders in the highly studied organisms Escherichia coli and Bacillus subtilis. Our results reveal several cases where multiple unrelated RNA structures likely bind the same r-protein ligand, and uncover previously unknown r-protein ligands. Each r-leader consistently occurs upstream of r-protein genes, suggesting a regulatory function. That the predicted r-leaders function as RNAs is supported by evolutionary correlations in the nucleotide sequences that are characteristic of a conserved RNA secondary structure. The r-leader predictions are also consistent with the locations of experimentally determined transcription start sites.ConclusionsThis work increases the number of known or predicted r-leader structures by more than 50%, providing additional opportunities to study structural and evolutionary aspects of RNA-protein interactions. These results provide a starting point for detailed experimental studies.

Estimating the power of sequence covariation for detecting conserved RNA structure.

Pairwise sequence covariations are a signal of conserved RNA secondary structure. We describe a method for distinguishing when lack of covariation signal can be taken as evidence against a conserved RNA structure, as opposed to when a sequence alignment merely has insufficient variation to detect covariations. We find that alignments for several long non-coding RNAs previously shown to lack covariation support do have adequate covariation detection power, providing additional evidence against their proposed conserved structures.Availability and implementationThe R-scape web server is at eddylab.org/R-scape, with a link to download the source code.Supplementary information Supplementary data are available at Bioinformatics online.

Biology and Function of miR159 in Plants

MicroR159 (miR159) is ancient, being present in the majority of land plants where it targets a class of regulatory genes called GAMYB or GAMYB-like via highly conserved miR159-binding sites. These GAMYB genes encode R2R3 MYB domain transcription factors that transduce the gibberellin (GA) signal in the seed aleurone and the anther tapetum. Here, GAMYB plays a conserved role in promoting the programmed cell death of these tissues, where miR159 function appears weak. By contrast, GAMYB is not involved in GA-signaling in vegetative tissues, but rather its expression is deleterious, leading to the inhibition of growth and development. Here, the major function of miR159 is to mediate strong silencing of GAMYB to enable normal growth. Highlighting this requirement of strong silencing are conserved RNA secondary structures associated with the miR159-binding site in GAMYB mRNA that promotes miR159-mediated repression. Although the miR159-GAMYB pathway in vegetative tissues has been implicated in a number of different functions, presently no conserved role for this pathway has emerged. We will review the current knowledge of the different proposed functions of miR159, and how this ancient pathway has been used as a model to help form our understanding of miRNA biology in plants.

Structural analyses of NEAT1 lncRNAs suggest long-range RNA interactions that may contribute to paraspeckle architecture.

Paraspeckles are nuclear bodies that regulate multiple aspects of gene expression. The long non-coding RNA (lncRNA) NEAT1 is essential for paraspeckle formation. NEAT1 has a highly ordered spatial organization within the paraspeckle, such that its 5′ and 3′ ends localize on the periphery of paraspeckle, while central sequences of NEAT1 are found within the paraspeckle core. As such, the structure of NEAT1 RNA may be important as a scaffold for the paraspeckle. In this study, we used SHAPE probing and computational analyses to investigate the secondary structure of human and mouse NEAT1. We propose a secondary structural model of the shorter (3,735 nt) isoform hNEAT1_S, in which the RNA folds into four separate domains. The secondary structures of mouse and human NEAT1 are largely different, with the exception of several short regions that have high structural similarity. Long-range base-pairing interactions between the 5′ and 3′ ends of the long isoform NEAT1 (NEAT1_L) were predicted computationally and verified using an in vitro RNA–RNA interaction assay. These results suggest that the conserved role of NEAT1 as a paraspeckle scaffold does not require extensively conserved RNA secondary structure and that long-range interactions among NEAT1 transcripts may have an important architectural function in paraspeckle formation.

Genome-wide identification of conserved intronic non-coding sequences using a Bayesian segmentation approach

BackgroundComputational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences.ResultsWe identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors.ConclusionsThis study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed.Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.

Search for 5'-leader regulatory RNA structures based on gene annotation aided by the RiboGap database.

The discovery of noncoding RNAs (ncRNAs) and their importance for gene regulation led us to develop bioinformatics tools to pursue the discovery of novel ncRNAs. Finding ncRNAs de novo is challenging, first due to the difficulty of retrieving large numbers of sequences for given gene activities, and second due to exponential demands on calculation needed for comparative genomics on a large scale. Recently, several tools for the prediction of conserved RNA secondary structure were developed, but many of them are not designed to uncover new ncRNAs, or are too slow for conducting analyses on a large scale. Here we present various approaches using the database RiboGap as a primary tool for finding known ncRNAs and for uncovering simple sequence motifs with regulatory roles. This database also can be used to easily extract intergenic sequences of eubacteria and archaea to find conserved RNA structures upstream of given genes. We also show how to extend analysis further to choose the best candidate ncRNAs for experimental validation.

A statistical test for conserved RNA structure shows lack of evidence for structure in lncRNAs.

Many functional RNAs have an evolutionarily conserved secondary structure. Conservation of RNA base pairing induces pairwise covariations in sequence alignments. We developed a computational method, R-scape (RNA Structural Covariation Above Phylogenetic Expectation), that quantitatively tests whether covariation analysis supports the presence of a conserved RNA secondary structure. R-scape analysis finds no statistically significant support for proposed secondary structures of the long noncoding RNAs HOTAIR, SRA, and Xist.

Corrigendum: Conserved RNA secondary structures and long-range interactions in hepatitis C viruses.

In the Discussion section (first paragraph, third sentence) on page 105 of the above-noted article, the sentence “The RNA architectures identified in our study were not predicted by in silico approaches (Pervouchine 2014), nor were they detected using recent high-throughput RNA structure sequencing techniques (Daines et al. 2011; Li et al. 2012)” was not clear and should instead read as follows: “The RNA bidirectional structural architectures identified in our study were not detected using recent high-throughput RNA structure sequencing techniques (Daines et al. 2011; Li et al. 2012), nor were they discovered by in silico approaches, although stem II of the Drosophila srp pre-mRNA was predicted (Raker et al. 2009; Pervouchine 2014).” Accordingly, the following reference should be included in the References section: Raker VA, Mironov AA, Gelfand MS, Pervouchine DD. 2009. Modulation of alternative splicing by long-range RNA structures in Drosophila. Nucleic Acids Res 37: 4533–4544. The authors apologize for any confusion this may have caused. This article has been corrected in both the PDF and full-text HTML files online.

Interval-Based Similarity for Classifying Conserved RNA Secondary Structures

The structure of a molecule is critical to determine its function in a biological context. Intelligent strategies are required to group structures with high similarity in an intuitive way. Most previous approaches focus on addressing sequence similarity and gene expression, whereas the techniques for comparative analysis of conserved structure data are underdeveloped because various secondary structures are complex, and most existing distance metrics have limitations. This article proposes a novel classification schema in terms of interval-based and weighted similarity functions that considers the intersection, non-intersection, and inclusion between two intervals of sequence size for conserved structures. The secondary structures are characterized by distance vectors. This assists in classifying structures under specific structure patterns that are expected to be correlated by functional or structural importance.

Prediction of Secondary Structures Conserved in Multiple RNA Sequences.

RNA structure is conserved by evolution to a greater extent than sequence. Predicting the conserved structure for multiple homologous sequences can be much more accurate than predicting the structure for a single sequence. RNAstructure is a software package that includes the programs Dynalign, Multilign, TurboFold, and PARTS for predicting conserved RNA secondary structure. This chapter provides protocols for using these programs.

A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.