Context-sensitivity of isosteric substitutions of non-Watson-Crick basepairs in recurrent RNA 3D motifs.

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.

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 .

Exploring Non-Coding RNAs in RNAcentral.

Non-coding RNAs are essential for all life and carry out a wide range of functions. Information about these molecules is distributed across dozens of specialized resources. RNAcentral is a database of non-coding RNA sequences that provides a unified access point to non-coding RNA annotations from >40 member databases and helps provide insight into the function of these RNAs. This article describes different ways of accessing the data, including searching the website and retrieving the data programmatically over web APIs and a public database. We also demonstrate an example Galaxy workflow for using RNAcentral for RNA-seq differential expression analysis. RNAcentral is available at https://rnacentral.org. © 2020 The Authors. Basic Protocol 1: Viewing RNAcentral sequence reports Basic Protocol 2: Using RNAcentral text search to explore ncRNA sequences Basic Protocol 3: Using RNAcentral sequence search Basic Protocol 4: Using RNAcentral FTP archive Support Protocol 1: Using web APIs for programmatic data access Support Protocol 2: Using public Postgres database to export large datasets Support Protocol 3: Analyze non-coding RNA in RNA-seq datasets using RNAcentral and Galaxy.

The RNA 3D Motif Atlas: Computational methods for extraction, organization and evaluation of RNA motifs.

RNA 3D motifs occupy places in structured RNA molecules that correspond to the hairpin, internal and multi-helix junction "loops" of their secondary structure representations. As many as 40% of the nucleotides of an RNA molecule can belong to these structural elements, which are distinct from the regular double helical regions formed by contiguous AU, GC, and GU Watson-Crick basepairs. With the large number of atomic- or near atomic-resolution 3D structures appearing in a steady stream in the PDB/NDB structure databases, the automated identification, extraction, comparison, clustering and visualization of these structural elements presents an opportunity to enhance RNA science. Three broad applications are: (1) identification of modular, autonomous structural units for RNA nanotechnology, nanobiology and synthetic biology applications; (2) bioinformatic analysis to improve RNA 3D structure prediction from sequence; and (3) creation of searchable databases for exploring the binding specificities, structural flexibility, and dynamics of these RNA elements. In this contribution, we review methods developed for computational extraction of hairpin and internal loop motifs from a non-redundant set of high-quality RNA 3D structures. We provide a statistical summary of the extracted hairpin and internal loop motifs in the most recent version of the RNA 3D Motif Atlas. We also explore the reliability and accuracy of the extraction process by examining its performance in clustering recurrent motifs from homologous ribosomal RNA (rRNA) structures. We conclude with a summary of remaining challenges, especially with regard to extraction of multi-helix junction motifs.

Identifying novel sequence variants of RNA 3D motifs.

Predicting RNA 3D structure from sequence is a major challenge in biophysics. An important sub-goal is accurately identifying recurrent 3D motifs from RNA internal and hairpin loop sequences extracted from secondary structure (2D) diagrams. We have developed and validated new probabilistic models for 3D motif sequences based on hybrid Stochastic Context-Free Grammars and Markov Random Fields (SCFG/MRF). The SCFG/MRF models are constructed using atomic-resolution RNA 3D structures. To parameterize each model, we use all instances of each motif found in the RNA 3D Motif Atlas and annotations of pairwise nucleotide interactions generated by the FR3D software. Isostericity relations between non-Watson-Crick basepairs are used in scoring sequence variants. SCFG techniques model nested pairs and insertions, while MRF ideas handle crossing interactions and base triples. We use test sets of randomly-generated sequences to set acceptance and rejection thresholds for each motif group and thus control the false positive rate. Validation was carried out by comparing results for four motif groups to RMDetect. The software developed for sequence scoring (JAR3D) is structured to automatically incorporate new motifs as they accumulate in the RNA 3D Motif Atlas when new structures are solved and is available free for download.

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.

An introduction to recurrent nucleotide interactions in RNA.

RNA secondary structure diagrams familiar to molecular biologists summarize at a glance the folding of RNA chains to form Watson­Crick paired double helices. However, they can be misleading: First of all, they imply that the nucleotides in loops and linker segments, which can amount to 35% to 50% of a structured RNA, do not significantly interact with other nucleotides. Secondly, they give the impression that RNA molecules are loosely organized in three-dimensional (3D) space. In fact, structured RNAs are compactly folded as a result of numerous long-range, sequence-specific interactions, many of which involve loop or linker nucleotides. Here, we provide an introduction for students and researchers of RNA on the types, prevalence, and sequence variations of inter-nucleotide interactions that structure and stabilize RNA 3D motifs and architectures, using Escherichia coli (E. coli) 16S ribosomal RNA as a concrete example. The picture that emerges is that almost all nucleotides in structured RNA molecules, including those in nominally single-stranded loop or linker regions, form specific interactions that stabilize functional structures or mediate interactions with other molecules. The small number of noninteracting, 'looped-out' nucleotides make it possible for the RNA chain to form sharp turns. Base-pairing is the most specific interaction in RNA as it involves edge-to-edge hydrogen bonding (H-bonding) of the bases. Non-Watson­Crick base pairs are a significant fraction (30% or more) of base pairs in structured RNAs.