Automatic detection of conserved RNA structure elements in complete RNA virus genomes.

We propose a new method for detecting conserved RNA secondary structures in a family of related RNA sequences. Our method is based on a combination of thermodynamic structure prediction and phylogenetic comparison. In contrast to purely phylogenetic methods, our algorithm can be used for small data sets of approximately 10 sequences, efficiently exploiting the information contained in the sequence variability. The procedure constructs a prediction only for those parts of sequences that are consistent with a single conserved structure. Our implementation produces reasonable consensus structures without user interference. As an example we have analysed the complete HIV-1 and hepatitis C virus (HCV) genomes as well as the small segment of hantavirus. Our method confirms the known structures in HIV-1 and predicts previously unknown conserved RNA secondary structures in HCV.     

Prediction of structured non-coding RNAs in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae

We present a survey for non-coding RNAs and other structured RNA motifs in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae using the RNAz program. This approach explicitly evaluates comparative sequence information to detect stabilizing selection acting on RNA secondary structure. We detect 3,672 structured RNA motifs, of which only 678 are known non-translated RNAs (ncRNAs) or clear homologs of known C. elegans ncRNAs. Most of these signals are located in introns or at a distance from known protein-coding genes. With an estimated false positive rate of about 50% and a sensitivity on the order of 50%, we estimate that the nematode genomes contain between 3,000 and 4,000 RNAs with evolutionary conserved secondary structures. Only a small fraction of these belongs to the known RNA classes, including tRNAs, snoRNAs, snRNAs, or microRNAs. A relatively small class of ncRNA candidates is associated with previously observed RNA-specific upstream elements.     

Secondary structure of the 5'' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.

The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.     

Predicted functional RNAs within coding regions constrain evolutionary rates of yeast proteins

Functional RNAs (fRNAs) are being recognized as an important regulatory component in biological processes. Interestingly, recent computational studies suggest that the number and biological significance of functional RNAs within coding regions (coding fRNAs) may have been underestimated. We hypothesized that such coding fRNAs will impose additional constraint on sequence evolution because the DNA primary sequence has to simultaneously code for functional RNA secondary structures on the messenger RNA in addition to the amino acid codons for the protein sequence. To test this prediction, we first utilized computational methods to predict conserved fRNA secondary structures within multiple species alignments of Saccharomyces sensu strico genomes. We predict that as much as 5% of the genes in the yeast genome contain at least one functional RNA secondary structure within their protein-coding region. We then analyzed the impact of coding fRNAs on the evolutionary rate of protein-coding genes because a decrease in evolutionary rate implies constraint due to biological functionality. We found that our predicted coding fRNAs have a significant influence on evolutionary rates (especially at synonymous sites), independent of other functional measures. Thus, coding fRNA may play a role on sequence evolution. Given that coding regions of humans and flies contain many more predicted coding fRNAs than yeast, the impact of coding fRNAs on sequence evolution may be substantial in genomes of higher eukaryotes.     

Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognized. We applied several computational methods to detect RNA secondary structure in the coding region of hepatitis C virus (HCV), including thermodynamic prediction, calculation of free energy on folding, and a newly developed method to scan sequences for covariant sites and associated secondary structures using a parsimony-based algorithm. Each of the prediction methods provided evidence for complex RNA folding in the core- and NS5B-encoding regions of the genome. The positioning of covariant sites and associated predicted stem-loop structures coincided with thermodynamic predictions of RNA base pairing, and localized precisely in parts of the genome with marked suppression of variability at synonymous sites. Combined, there was evidence for a total of six evolutionarily conserved stem-loop structures in the NS5B-encoding region and two in the core gene. The virus most closely related to HCV, GB virus-B (GBV-B) also showed evidence for similar internal base pairing in its coding region, although predictions of secondary structures were limited by the absence of comparative sequence data for this virus. While the role(s) of stem-loops in the coding region of HCV and GBV-B are currently unknown, the structure predictions in this study could provide the starting point for functional investigations using recently developed self-replicating clones of HCV.     

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.     

Role of the hepatitis C virus core+1 open reading frame and core cis-acting RNA elements in viral RNA translation and replication

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.     

Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus.     

Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1_NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.     

Structural and functional analysis of the RNA transport element, a member of an extensive family present in the mouse genome

We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol. 75:4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected Xenopus laevis oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s).     

Endogenous viral elements in animal genomes

Integration into the nuclear genome of germ line cells can lead to vertical inheritance of retroviral genes as host alleles. For other viruses, germ line integration has only rarely been documented. Nonetheless, we identified endogenous viral elements (EVEs) derived from ten non-retroviral families by systematic in silico screening of animal genomes, including the first endogenous representatives of double-stranded RNA, reverse-transcribing DNA, and segmented RNA viruses, and the first endogenous DNA viruses in mammalian genomes. Phylogenetic and genomic analysis of EVEs across multiple host species revealed novel information about the origin and evolution of diverse virus groups. Furthermore, several of the elements identified here encode intact open reading frames or are expressed as mRNA. For one element in the primate lineage, we provide statistically robust evidence for exaptation. Our findings establish that genetic material derived from all known viral genome types and replication strategies can enter the animal germ line, greatly broadening the scope of paleovirological studies and indicating a more significant evolutionary role for gene flow from virus to animal genomes than has previously been recognized.     

An evolutionary model for protein-coding regions with conserved RNA structure

Here we present a model of nucleotide substitution in protein-coding regions that also encode the formation of conserved RNA structures. In such regions, apparent evolutionary context dependencies exist, both between nucleotides occupying the same codon and between nucleotides forming a base pair in the RNA structure. The overlap of these fundamental dependencies is sufficient to cause "contagious" context dependencies which cascade across many nucleotide sites. Such large-scale dependencies challenge the use of traditional phylogenetic models in evolutionary inference because they explicitly assume evolutionary independence between short nucleotide tuples. In our model we address this by replacing context dependencies within codons by annotation-specific heterogeneity in the substitution process. Through a general procedure, we fragment the alignment into sets of short nucleotide tuples based on both the protein coding and the structural annotation. These individual tuples are assumed to evolve independently, and the different tuple sets are assigned different annotation-specific substitution models shared between their members. This allows us to build a composite model of the substitution process from components of traditional phylogenetic models. We applied this to a data set of full-genome sequences from the hepatitis C virus where five RNA structures are mapped within the coding region. This allowed us to partition the effects of selection on different structural elements and to test various hypotheses concerning the relation of these effects. Of particular interest, we found evidence of a functional role of loop and bulge regions, as these were shown to evolve according to a different and more constrained selective regime than the nonpairing regions outside the RNA structures. Other potential applications of the model include comparative RNA structure prediction in coding regions and RNA virus phylogenetics.     

Conserved secondary structures in Aspergillus

Background

Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs ) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs.

Methodology/Principal Findings

We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted ∼60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 5′ UTRs primarily contain groupings of short hairpins located near the start codon, and 3′ UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene.

Conclusions/Sigificance

There are many conserved, high-confidence RNAs of unknown function in these Aspergillus genomes, as well as interesting spatial distributions of predicted secondary structures. This study increases our knowledge of secondary structure in these aspergillus organisms.     

A novel method for the identification of conserved structural patterns in RNA: From small scale to high-throughput applications

Functional RNA regions are often related to recurrent secondary structure patterns (or motifs), which can exert their role in several different ways, particularly in dictating the interaction with RNA-binding proteins, and acting in the regulation of a large number of cellular processes. Among the available motif-finding tools, the majority focuses on sequence patterns, sometimes including secondary structure as additional constraints to improve their performance. Nonetheless, secondary structures motifs may be concurrent to their sequence counterparts or even encode a stronger functional signal. Current methods for searching structural motifs generally require long pipelines and/or high computational efforts or previously aligned sequences. Here, we present BEAM (BEAr Motif finder), a novel method for structural motif discovery from a set of unaligned RNAs, taking advantage of a recently developed encoding for RNA secondary structure named BEAR (Brand nEw Alphabet for RNAs) and of evolutionary substitution rates of secondary structure elements. Tested in a varied set of scenarios, from small- to large-scale, BEAM is successful in retrieving structural motifs even in highly noisy data sets, such as those that can arise in CLIP-Seq or other high-throughput experiments.     

RNA folding: pseudoknots, loops and bulges

The three-dimensional structures adopted by RNA molecules are crucial to their biological functions. The nucleotides of an RNA molecule interact to form characteristic secondary-structure motifs. Tertiary interactions orient these secondary-structure elements with respect to each other to form the functional RNA. Here we describe the basic structural elements with special emphasis on a novel tertiary motif, the pseudoknot.     

Comparison of SIV and HIV-1 Genomic RNA Structures Reveals Impact of Sequence Evolution on Conserved and Non-Conserved Structural Motifs

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1_NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1_NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1_NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve.     

Common structures of the 5' non-coding RNA in enteroviruses and rhinoviruses. Thermodynamical stability and statistical significance

A total of 4051 suboptimal secondary structures are predicted by folding the 5' non-coding region of ten polioviruses, five human rhinoviruses and three coxsackieviruses using our new suboptimal folding algorithm for the prediction of both optimal and suboptimal RNA secondary structures. A comparative analysis of these RNA secondary structures reveals the conservation of common secondary structure that can be supported by phylogenetic data. The thermodynamic stability and statistical significance of these predicted, conserved helical elements are assessed and significant structure motifs in the 5' non-coding region are proposed. The possible roles of these structure motifs in the virus life cycle are discussed.     

Cis-acting RNA elements in human and animal plus-strand RNA viruses

The RNA genomes of plus-strand RNA viruses have the ability to form secondary and higher-order structures that contribute to their stability and to their participation in inter- and intramolecular interactions. Those structures that are functionally important are called cis-acting RNA elements because their functions cannot be complemented in trans. They can be involved not only in RNA/RNA interactions but also in binding of viral and cellular proteins during the complex processes of translation, RNA replication and encapsidation. Most viral cis-acting RNA elements are located in the highly structured 5′- and 3′-nontranslated regions of the genomes but sometimes they also extend into the adjacent coding sequences. In addition, some cis-acting RNA elements are embedded within the coding sequences far away from the genomic ends. Although the functional importance of many of these structures has been confirmed by genetic and biochemical analyses, their precise roles are not yet fully understood. In this review we have summarized what is known about cis-acting RNA elements in nine families of human and animal plus-strand RNA viruses with an emphasis on the most thoroughly characterized virus families, the Picornaviridae and Flaviviridae.     

Detection of tRNA-like structure through RNase P cleavage of viral internal ribosome entry site RNAs near the AUG start triplet

The 9600-base RNA genome of hepatitis C virus (HCV) has an internal ribosome entry site (IRES) in its first 370 bases, including the AUG start triplet at bases 342-344. Structural elements of this and other IRES domains substitute for a 5' terminal cap structure in protein synthesis. Recent work (Nadal, A., Martell, M., Lytle, J. R., Lyons, A. J., Robertson, H. D., Cabot, B., Esteban, J. I., Esteban, R., Guardia, J., and Gomez, J. (2002) J. Biol. Chem. 277, 30606-30613) has demonstrated that the host pre-tRNA processing enzyme, RNase P, can cleave the HCV RNA genome at a site in the IRES near the AUG initiator triplet. Although this step is unlikely to be part of the HCV life cycle, such a reaction could indicate the presence of a tRNA-like structure in this IRES. Because susceptibility to cleavage by mammalian RNase P is a strong indicator of tRNA-like structure, we have conducted the studies reported here to test whether such tRNA mimicry is unique to HCV or is a general property of IRES structure. We have assayed IRES domains of several viral RNA genomes: two pestiviruses related to HCV, classical swine fever virus and bovine viral diarrhea virus; and two unrelated viruses, encephalomyocarditis virus and cricket paralysis virus. We have found similarly placed RNase P cleavage sites in these IRESs. Thus a tRNA-like domain could be a general structural feature of IRESs, the first IRES structure to be identified with a functional correlate. Such tRNA-like features could be recognized by pre-existing ribosomal tRNA-binding sites as part of the IRES initiation cycle.