RNAcentral: a comprehensive database of non-coding RNA sequences.

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.

R3D-2-MSA: the RNA 3D structure-to-multiple sequence alignment server.

The RNA 3D Structure-to-Multiple Sequence Alignment Server (R3D-2-MSA) is a new web service that seamlessly links RNA three-dimensional (3D) structures to high-quality RNA multiple sequence alignments (MSAs) from diverse biological sources. In this first release, R3D-2-MSA provides manual and programmatic access to curated, representative ribosomal RNA sequence alignments from bacterial, archaeal, eukaryal and organellar ribosomes, using nucleotide numbers from representative atomic-resolution 3D structures. A web-based front end is available for manual entry and an Application Program Interface for programmatic access. Users can specify up to five ranges of nucleotides and 50 nucleotide positions per range. The R3D-2-MSA server maps these ranges to the appropriate columns of the corresponding MSA and returns the contents of the columns, either for display in a web browser or in JSON format for subsequent programmatic use. The browser output page provides a 3D interactive display of the query, a full list of sequence variants with taxonomic information and a statistical summary of distinct sequence variants found. The output can be filtered and sorted in the browser. Previous user queries can be viewed at any time by resubmitting the output URL, which encodes the search and re-generates the results. The service is freely available with no login requirement at http://rna.bgsu.edu/r3d-2-msa.

Specificity between lactobacilli and hymenopteran hosts is the exception rather than the rule.

Lactobacilli (Lactobacillales: Lactobacillaceae) are well known for their roles in food fermentation, as probiotics, and in human health, but they can also be dominant members of the microbiota of some species of Hymenoptera (ants, bees, and wasps). Honey bees and bumble bees associate with host-specific lactobacilli, and some evidence suggests that these lactobacilli are important for bee health. Social transmission helps maintain associations between these bees and their respective microbiota. To determine whether lactobacilli associated with social hymenopteran hosts are generally host specific, we gathered publicly available Lactobacillus 16S rRNA gene sequences, along with Lactobacillus sequences from 454 pyrosequencing surveys of six other hymenopteran species (three sweat bees and three ants). We determined the comparative secondary structural models of 16S rRNA, which allowed us to accurately align the entire 16S rRNA gene, including fast-evolving regions. BLAST searches and maximum-likelihood phylogenetic reconstructions confirmed that honey and bumble bees have host-specific Lactobacillus associates. Regardless of colony size or within-colony oral sharing of food (trophallaxis), sweat bees and ants associate with lactobacilli that are closely related to those found in vertebrate hosts or in diverse environments. Why honey and bumble bees associate with host-specific lactobacilli while other social Hymenoptera do not remains an open question. Lactobacilli are known to inhibit the growth of other microbes and can be beneficial whether they are coevolved with their host or are recruited by the host from environmental sources through mechanisms of partner choice.

R2DT is a framework for predicting and visualising RNA secondary structure using templates.

Non-coding RNAs (ncRNA) are essential for all life, and their functions often depend on their secondary (2D) and tertiary structure. Despite the abundance of software for the visualisation of ncRNAs, few automatically generate consistent and recognisable 2D layouts, which makes it challenging for users to construct, compare and analyse structures. Here, we present R2DT, a method for predicting and visualising a wide range of RNA structures in standardised layouts. R2DT is based on a library of 3,647 templates representing the majority of known structured RNAs. R2DT has been applied to ncRNA sequences from the RNAcentral database and produced >13 million diagrams, creating the world's largest RNA 2D structure dataset. The software is amenable to community expansion, and is freely available at https://github.com/rnacentral/R2DT and a web server is found at https://rnacentral.org/r2dt .

Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes.

BACKGROUND: Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. RESULTS: In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. CONCLUSIONS: Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

Structure of the mammalian 80S ribosome at 8.7 A resolution.

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.

Supersized Ribosomal RNA Expansion Segments in Asgard Archaea.

The ribosome's common core, comprised of ribosomal RNA (rRNA) and universal ribosomal proteins, connects all life back to a common ancestor and serves as a window to relationships among organisms. The rRNA of the common core is similar to rRNA of extant bacteria. In eukaryotes, the rRNA of the common core is decorated by expansion segments (ESs) that vastly increase its size. Supersized ESs have not been observed previously in Archaea, and the origin of eukaryotic ESs remains enigmatic. We discovered that the large ribosomal subunit (LSU) rRNA of two Asgard phyla, Lokiarchaeota and Heimdallarchaeota, considered to be the closest modern archaeal cell lineages to Eukarya, bridge the gap in size between prokaryotic and eukaryotic LSU rRNAs. The elongated LSU rRNAs in Lokiarchaeota and Heimdallarchaeota stem from two supersized ESs, called ES9 and ES39. We applied chemical footprinting experiments to study the structure of Lokiarchaeota ES39. Furthermore, we used covariation and sequence analysis to study the evolution of Asgard ES39s and ES9s. By defining the common eukaryotic ES39 signature fold, we found that Asgard ES39s have more and longer helices than eukaryotic ES39s. Although Asgard ES39s have sequences and structures distinct from eukaryotic ES39s, we found overall conservation of a three-way junction across the Asgard species that matches eukaryotic ES39 topology, a result consistent with the accretion model of ribosomal evolution.

The limits of nuclear encoded SSU rDNA for resolving the diatom phylogeny.

A recent reclassification of diatoms based on phylogenies recovered using the nuclear-encoded SSU rRNA gene contains three major classes, Coscinodiscophyceae, Mediophyceae and the Bacillariophyceae (the CMB hypothesis). We evaluated this with a sequence alignment of 1336 protist and heterokont algae SSU rRNAs, which includes 673 diatoms. Sequences were aligned to maintain structural elements conserved within this dataset. Parsimony analysis rejected the CMB hypothesis, albeit weakly. Morphological data are also incongruent with this recent CMB hypothesis of three diatom clades. We also reanalyzed a recently published dataset which purports to support the CMB hypothesis. Our reanalysis found that the original analysis had not converged on the true bipartition posterior probability distribution, and rejected the CMB hypothesis. Thus we conclude that a reclassification of the evolutionary relationships of the diatoms according to the CMB hypothesis is premature.

The accuracy of ribosomal RNA comparative structure models.

The determination of the 16S and 23S rRNA secondary structure models was initiated shortly after the first complete 16S and 23S rRNA sequences were determined in the late 1970s. The structures that are common to all 16S rRNAs and all 23S rRNAs were determined using comparative methods from the analysis of thousands of rRNA sequences. Twenty-plus years later, the 16S and 23S rRNA comparative structure models have been evaluated against the recently determined high-resolution crystal structures of the 30S and 50S ribosomal subunits. Nearly all of the predicted covariation-based base pairs, including the regular base pairs and helices, and the irregular base pairs and tertiary interactions, were present in the 30S and 50S crystal structures.

The lonepair triloop: a new motif in RNA structure.

The lonepair triloop (LPTL) is an RNA structural motif that contains a single ("lone") base-pair capped by a hairpin loop containing three nucleotides. The two nucleotides immediately outside of this motif (5' and 3' to the lonepair) are not base-paired to one another, restricting the length of this helix to a single base-pair. Four examples of this motif, along with three tentative examples, were initially identified in the 16S and 23S rRNAs with covariation analysis. An evaluation of the recently determined crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits revealed the authenticity for all of these proposed interactions and identified 16 more LPTLs in the 5S, 16S and 23S rRNAs. This motif is found in the T loop in the tRNA crystal structures. The lonepairs are positioned, in nearly all examples, immediately 3' to a regular secondary structure helix and are stabilized by coaxial stacking onto this flanking helix. In all but two cases, the nucleotides in the triloop are involved in a tertiary interaction with another section of the rRNA, establishing an overall three-dimensional function for this motif. Of these 24 examples, 14 occur in multi-stem loops, seven in hairpin loops and three in internal loops. While the most common lonepair, U:A, occurs in ten of the 24 LPTLs, the remaining 14 LPTLs contain seven different base-pair types. Only a few of these lonepairs adopt the standard Watson-Crick base-pair conformations, while the majority of the base-pairs have non-standard conformations. While the general three-dimensional conformation is similar for all examples of this motif, characteristic differences lead to several subtypes present in different structural environments. At least one triloop nucleotide in 22 of the 24 LPTLs in the rRNAs and tRNAs forms a tertiary interaction with another part of the RNA. When a LPTL containing the GNR or UYR triloop sequence forms a tertiary interaction with the first (and second) triloop nucleotide, it recruits a fourth nucleotide to mediate stacking and mimic the tetraloop conformation. Approximately half of the LPTL motifs are in close association with proteins. The majority of these LPTLs are positioned at sites in rRNAs that are conserved in the three phylogenetic domains; a few of these occur in regions of the rRNA associated with ribosomal function, including the presumed site of peptidyl transferase activity in the 23S rRNA.

Modeling a minimal ribosome based on comparative sequence analysis.

We have determined the three-dimensional organization of ribosomal RNAs and proteins essential for minimal ribosome function. Comparative sequence analysis identifies regions of the ribosome that have been evolutionarily conserved, and the spatial organization of conserved domains is determined by mapping these onto structures of the 30S and 50S subunits determined by X-ray crystallography. Several functional domains of the ribosome are conserved in their three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplast ribosomes. In contrast, other regions from both subunits have shifted their position in three-dimensional space during evolution, including the L11 binding domain and the alpha-sarcin-ricin loop (SRL). We examined conserved bridge interactions between the two ribosomal subunits, giving an indication of which contacts are more significant. The tRNA contacts that are conserved were also determined, highlighting functional interactions as the tRNA moves through the ribosome during protein synthesis. To augment these studies of a large collection of comparative structural models sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans mitochondrial rRNA is considered individually because it is among the smallest rRNA sequences known. The C.elegans model supports the large collection of comparative structure models while providing insight into the evolution of mitochondrial ribosomes.

Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction.

BACKGROUND: A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1. RESULTS: The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases. CONCLUSION: Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.

The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs.

BACKGROUND: Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. RESULTS: We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. CONCLUSIONS: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.

The exon context and distribution of Euascomycetes rRNA spliceosomal introns.

BACKGROUND: We have studied spliceosomal introns in the ribosomal (r)RNA of fungi to discover the forces that guide their insertion and fixation. RESULTS: Comparative analyses of flanking sequences at 49 different spliceosomal intron sites showed that the G - intron - G motif is the conserved flanking sequence at sites of intron insertion. Information analysis showed that these rRNA introns contain significant information in the flanking exons. Analysis of all rDNA introns in the three phylogenetic domains and two organelles showed that group I introns are usually located after the most conserved sites in rRNA, whereas spliceosomal introns occur at less conserved positions. The distribution of spliceosomal and group I introns in the primary structure of small and large subunit rRNAs was tested with simulations using the broken-stick model as the null hypothesis. This analysis suggested that the spliceosomal and group I intron distributions were not produced by a random process. Sequence upstream of rRNA spliceosomal introns was significantly enriched in G nucleotides. We speculate that these G-rich regions may function as exonic splicing enhancers that guide the spliceosome and facilitate splicing. CONCLUSIONS: Our results begin to define some of the rules that guide the distribution of rRNA spliceosomal introns and suggest that the exon context is of fundamental importance in intron fixation.

Two accurate sequence, structure, and phylogenetic template-based RNA alignment systems.

BACKGROUND: The analysis of RNA sequences, once a small niche field for a small collection of scientists whose primary emphasis was the structure and function of a few RNA molecules, has grown most significantly with the realizations that 1) RNA is implicated in many more functions within the cell, and 2) the analysis of ribosomal RNA sequences is revealing more about the microbial ecology within all biological and environmental systems. The accurate and rapid alignment of these RNA sequences is essential to decipher the maximum amount of information from this data. METHODS: Two computer systems that utilize the Gutell lab's RNA Comparative Analysis Database (rCAD) were developed to align sequences to an existing template alignment available at the Gutell lab's Comparative RNA Web (CRW) Site. Multiple dimensions of cross-indexed information are contained within the relational database--rCAD, including sequence alignments, the NCBI phylogenetic tree, and comparative secondary structure information for each aligned sequence. The first program, CRWAlign-1 creates a phylogenetic-based sequence profile for each column in the alignment. The second program, CRWAlign-2 creates a profile based on phylogenetic, secondary structure, and sequence information. Both programs utilize their profiles to align new sequences into the template alignment. RESULTS: The accuracies of the two CRWAlign programs were compared with the best template-based rRNA alignment programs and the best de-novo alignment programs. We have compared our programs with a total of eight alternative alignment methods on different sets of 16S rRNA alignments with sequence percent identities ranging from 50% to 100%. Both CRWAlign programs were superior to these other programs in accuracy and speed. CONCLUSIONS: Both CRWAlign programs can be used to align the very extensive amount of RNA sequencing that is generated due to the rapid next-generation sequencing technology. This latter technology is augmenting the new paradigm that RNA is intimately implicated in a significant number of functions within the cell. In addition, the use of bacterial 16S rRNA sequencing in the identification of the microbiome in many different environmental systems creates a need for rapid and highly accurate alignment of bacterial 16S rRNA sequences.

An Accurate Scalable Template-based Alignment Algorithm.

The rapid determination of nucleic acid sequences is increasing the number of sequences that are available. Inherent in a template or seed alignment is the culmination of structural and functional constraints that are selecting those mutations that are viable during the evolution of the RNA. While we might not understand these structural and functional, template-based alignment programs utilize the patterns of sequence conservation to encapsulate the characteristics of viable RNA sequences that are aligned properly. We have developed a program that utilizes the different dimensions of information in rCAD, a large RNA informatics resource, to establish a profile for each position in an alignment. The most significant include sequence identity and column composition in different phylogenetic taxa. We have compared our methods with a maximum of eight alternative alignment methods on different sets of 16S and 23S rRNA sequences with sequence percent identities ranging from 50% to 100%. The results showed that CRWAlign outperformed the other alignment methods in both speed and accuracy. A web-based alignment server is available at http://www.rna.ccbb.utexas.edu/SAE/2F/CRWAlign.

The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum.

BACKGROUND: The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. PRINCIPAL FINDINGS: The identification of 14 additional small mitochondrial transcripts from P. falciparum and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. SIGNIFICANCE: All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.

RNA2DMap: A Visual Exploration Tool of the Information in RNA's Higher-Order Structure.

A new and emerging paradigm in molecular biology is revealing that RNA is implicated in nearly every aspect of the metabolism in the cell. To enhance our understanding of the function of these RNA molecules in the cell, it is essential that we have a complete understanding of their higher-order structures. While many computational tools have been developed to predict and analyse these higher-order RNA structures, few are able to visualize them for analytical purposes. In this paper, we present an interactive visualization tool of the secondary structure of RNA, named RNA2DMap. This program enables multiple-dimensions of information about RNA structure to be selected, customized and displayed to visually identify patterns and relationships. RNA2DMap facilitates the comparative analysis and understanding of RNAs that cannot be readily obtained with other graphical or text output from computer programs. Three use cases are presented to illustrate how RNA2DMap aids structural analysis.

ITS secondary structure derived from comparative analysis: implications for sequence alignment and phylogeny of the Asteraceae.

An RNA secondary structure model is presented for the nuclear ribosomal internal transcribed spacers (ITS) based on comparative analysis of 340 sequences from the angiosperm family Asteraceae. The model based on covariation analysis agrees with structural features proposed in previous studies using mainly thermodynamic criteria and provides evidence for additional structural motifs within ITS1 and ITS2. The minimum structure model suggests that at least 20% of ITS1 and 38% of ITS2 nucleotide positions are involved in base pairing to form helices. The sequence alignment enabled by conserved structural features provides a framework for broadscale molecular evolutionary studies and the first family-level phylogeny of the Asteraceae based on nuclear DNA data. The phylogeny based on ITS sequence data is very well resolved and shows considerable congruence with relationships among major lineages of the family suggested by chloroplast DNA studies, including a monophyletic subfamily Asteroideae and a paraphyletic subfamily Cichorioideae. Combined analyses of ndhF and ITS sequences provide additional resolution and support for relationships in the family.