Large RNA Structures Research Articles

Structure and genome organization of the large RNA of iris yellow spot virus (genus Tospovirus, family Bunyaviridae)

The structure and organization of the large (L) RNA of iris yellow spot virus (IYSV) was determined, and with this report, the complete genomic sequence of IYSV of the genus Tospovirus, family Bunyaviridae has been elucidated. The L RNA of IYSV was 8,880 nucleotides in length and contained a single open reading frame in the viral complementary (vc) strand. The primary translation product of 331.17 kDa shared many of the features of the viral RNA-dependent RNA polymerase (RdRp) coded by L RNAs of known tospoviruses. The 5' and 3' termini of IYSV L RNA (vc) contain two untranslated regions of 33 and 226 nucleotides, respectively, and both termini have conserved terminal nucleotides, another common feature of tospovirus genomic RNAs. Conserved motifs characteristic of RdRps of members of the family Bunyaviridae were present in the IYSV RdRp.

PseudoViewer3: generating planar drawings of large-scale RNA structures with pseudoknots

Pseudoknots in RNA structures make visualization of RNA structures difficult. Even if a pseudoknot itself is represented without a crossing, visualization of the entire RNA structure with a pseudoknot often results in a drawing with crossings between the pseudoknot and other structural elements, and requires additional intervention by the user to ensure that the structure graph is overlap-free. Many programs such as web services prefer to obtain an overlap-free graph in one-shot rather than get a graph with overlaps to be edited. There are few programs for visualizing RNA pseudoknots, and PseudoViewer has been the almost only program that automatically draws RNA secondary structures with pseudoknots. The previous version of PseudoViewer visualizes all the known types of RNA pseudoknots as planar drawings, but visualizes some hypothetical pseudoknots as non-planar drawings. We developed a new version of PseudoViewer for efficiently visualizing large RNA structures with any types of pseudoknots, both known and hypothetical, as planar drawings in one-shot. It is about 10 times faster than the previous algorithm, and produces a more compact and aesthetic structure drawing. PseudoViewer3 supports both web services and web applications. The new version of PseudoViewer, PseudoViewer3, is available at (http://pseudoviewer.inha.ac.kr).

Finding 3D motifs in ribosomal RNA structures

The identification of small structural motifs and their organization into larger subassemblies is of fundamental interest in the analysis, prediction and design of 3D structures of large RNAs. This problem has been studied only sparsely, as most of the existing work is limited to the characterization and discovery of motifs in RNA secondary structures. We present a novel geometric method for the characterization and identification of structural motifs in 3D rRNA molecules. This method enables the efficient recognition of known 3D motifs, such as tetraloops, E-loops, kink-turns and others. Furthermore, it provides a new way of characterizing complex 3D motifs, notably junctions, that have been defined and identified in the secondary structure but have not been analyzed and classified in three dimensions. We demonstrate the relevance and utility of our approach by applying it to the Haloarcula marismortui large ribosomal unit. Pending the implementation of a dedicated web server, the code accompanying this article, written in JAVA, is available upon request from the contact author.

Sequence to Structure (S2S): display, manipulate and interconnect RNA data from sequence to structure

Efficient RNA sequence manipulations (such as multiple alignments) need to be constrained by rules of RNA structure folding. The structural knowledge has increased dramatically in the last years with the accumulation of several large RNA structures similar to those of the bacterial ribosome subunits. However, no tool in the RNA community provides an easy way to link and integrate progress made at the sequence level using the available three-dimensional information. Sequence to Structure (S2S) proposes a framework in which an user can easily display, manipulate and interconnect heterogeneous RNA data, such as multiple sequence alignments, secondary and tertiary structures. S2S has been implemented using the Java language and has been developed and tested under UNIX systems, such as Linux and MacOSX. S2S is available at http://bioinformatics.org/S2S/.

A method for aligning RNA secondary structures and its application to RNA motif detection

BackgroundAlignment of RNA secondary structures is important in studying functional RNA motifs. In recent years, much progress has been made in RNA motif finding and structure alignment. However, existing tools either require a large number of prealigned structures or suffer from high time complexities. This makes it difficult for the tools to process RNAs whose prealigned structures are unavailable or process very large RNA structure databases.ResultsWe present here an efficient tool called RSmatch for aligning RNA secondary structures and for motif detection. Motivated by widely used algorithms for RNA folding, we decompose an RNA secondary structure into a set of atomic structure components that are further organized by a tree model to capture the structural particularities. RSmatch can find the optimal global or local alignment between two RNA secondary structures using two scoring matrices, one for single-stranded regions and the other for double-stranded regions. The time complexity of RSmatch is O(mn) where m is the size of the query structure and n that of the subject structure. When applied to searching a structure database, RSmatch can find similar RNA substructures, and is capable of conducting multiple structure alignment and iterative database search. Therefore it can be used to identify functional RNA motifs. The accuracy of RSmatch is tested by experiments using a number of known RNA structures, including simple stem-loops and complex structures containing junctions.ConclusionWith respect to computing efficiency and accuracy, RSmatch compares favorably with other tools for RNA structure alignment and motif detection. This tool shall be useful to researchers interested in comparing RNA structures obtained from wet lab experiments or RNA folding programs, particularly when the size of the structure dataset is large.

Calculation of Standard Atomic Volumes for RNA and Comparison with Proteins: RNA is Packed More Tightly

Traditionally, for biomolecular packing calculations research has focused on proteins. Besides proteins, RNA is the other large biomolecule that has tertiary structure interactions and complex packing. No one has yet quantitatively investigated RNA packing or compared its packing to that of proteins because, until recently, there were no large RNA structures. Here we address this question in detail, using Voronoi volume calculations on a set of high-resolution RNA crystal structures. We do a careful parameterization, taking into account many factors such as atomic radii, crystal packing, structural complexity, solvent, and associated protein to obtain a self-consistent, universal set of volumes that can be applied to both RNA and protein. We report this set of volumes, which we call the NucProt parameter set. Our measured values are consistent across the many different RNA structures and packing environments. When common atom types are compared between proteins and RNA, nine of 12 types show that RNA has a smaller volume and packs more tightly than protein, suggesting that close-packing may be as important for the folding of RNAs as it is for proteins. Moreover, calculated partial specific volumes show that RNA bases pack more densely than corresponding aromatic residues from proteins. Finally, we find that RNA bases have similar packing volumes to DNA bases, despite the absence of tertiary contacts in DNA. Programs, parameter sets and raw data are available online at.

Site‐Specific Fluorescent Labeling of Large RNAs with Pyrene

Pyrene is a useful chromophore for monitoring the tertiary structure and folding of large RNAs. This unit describes the general preparation of a large RNA (>80 nucleotides in length) that has been site-specifically modified with pyrene at the 2'-position of an individual internal nucleotide. A protocol is provided for derivatizing a 2'-amino-RNA oligonucleotide with a suitably activated pyrene reagent. This pyrene-labeled oligonucleotide is then assembled with other RNA(s) either by covalent ligation or by noncovalent hybridization to form a full-length structured RNA, which may then be studied by equilibrium and stopped-flow fluorescence spectroscopy.

Spectral Decomposition for the Search and Analysis of RNA Secondary Structure

Scales in RNA, based on geometrical considerations, can be exploited for the analysis and prediction of RNA structures. By using spectral decomposition, geometric information that relates to a given RNA fold can be reduced to a single positive scalar number, the second eigenvalue of the Laplacian matrix corresponding to the tree-graph representation of the RNA secondary structure. Along with the free energy of the structure, being the most important scalar number in the prediction of RNA folding by energy minimization methods, the second eigenvalue of the Laplacian matrix can be used as an effective signature for locating a target folded structure given a set of RNA folds. Furthermore, the second eigenvector of the Laplacian matrix can be used to partition large RNA structures into smaller fragments. An illustrative example is given for the use of the second eigenvalue to predict mutations that may cause structural rearrangements, thereby disrupting stable motifs.

Evidence from the small and large ribosomal RNA structure suggests that Anoplostoma rectospiculum Gal'tsova, 1976 (Nematoda: Anoplostomatidae) is a member of the superfamily Enoploidea, not Oncholaimoidea

Abstract Analyses of the primary structure of the 18S rRNA gene and D3 segment of the 28S rRNA, as well as evidence from the secondary structure of the SSU rRNA V7 region, suggest that Anoplostoma rectospiculum (Anoplostomatidae) has closer relationships to the family Enoplidae than to the Oncholaimidae. In phylogenetic trees derived from full length SSU rRNA gene and partial LSU rRNA gene (D3 expansion segment) sequence analyses, A. rectospiculum exhibits long branches. The associated artefacts of long branch attraction (LBA) are circumvented because of the presence of an undoubted molecular synapomorphy – a low homoplastic 1. b. p. insertion in helix 43 of the SSU rRNA which is shared jointly by Anoplostomatidae and Enoplidae. Analysis of low homoplastic apomorphic characters is considered to be a tool for testing phylogenies against LBA artefacts.

Structure of the Pyrimidine-rich Internal Loop in the Poliovirus 3′-UTR: The Importance of Maintaining Pseudo-2-fold Symmetry in RNA Helices Containing Two Adjacent Non-canonical Base-pairs

Formation of non-canonical base-pairs in RNA often plays a very important functional role. In addition they frequently serve as factors in stabilizing the secondary structure elements that provide the frame of large compact RNA structures. Here we describe the structure of an internal loop containing a 5′CU3′/5′UU3′ non-canonical tandem base-pair motif, which is conserved within the 3′-UTR of poliovirus-like enteroviruses. Structural details reveal striking regularities of the local helix geometry, resulting from alternating geometrical adjustments, which are important for understanding and predicting stabilities and configurations of tandem non-canonical base-pairs. The C-U and U-U base-pairs severely contract the minor groove of the sugar-phosphate backbone, which might be important for protein recognition or binding to other RNA elements.

RNA tertiary interactions in the large ribosomal subunit: the A-minor motif.

Analysis of the 2.4-A resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2' OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3' terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

12] Solving large RNA structures by X-ray crystallography

This chapter discusses the application of X-ray crystallography for solving large RNA structures. RNA crystals tend to decay rapidly when exposed to X rays. Thus, diffraction data are generally collected from RNA crystals at cryogenic temperatures. Compounds used in protein crystal stabilization are useful for RNA crystals as well. These include sugars and nonvolatile alcohols, such as glucose, glycerol, 2-methyl-2,4-pentanediol (MPD), and low molecular weight polyethylene glycols (PEGs). In addition, volatile alcohols, such as ethanol and isopropanol, have been useful in RNA crystal cryostabilization. Typically, the cryoprotectant should constitute at least 15–20% (v/v) of the soaking solution for the crystals. Addition of the cryoprotectant should be in 1–2% increments by adding solution to the drop containing the crystal and resealing the coverslip to prevent evaporation. If the crystal appears unchanged after 2 minutes, another addition of the protectant can be made. Once the final concentration of cryoprotectant in the drop has been adjusted to high enough concentrations, the reservoir solution within the crystallization setup should be adjusted to the same composition and the system resealed to equilibrate. Two kinds of transition metal ions have been used with great success in RNA crystallography. First, lanthanide series cations contributed to solving the structures of tRNA, the hammerhead ribozyme, and the group I intron P4–P6 domain. Second, osmium(III) hexammine provided the high-resolution phasing for the P4–P6 domain structure.

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns.

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.

A pseudoknot-compatible universal site is located in the large ribosomal RNA in the peptidyltransferase center

The RNA secondary structure is not confined to a system of the hairpins and can contain pseudoknots as well as topologically equivalent slipped-loop structure (SLS) conformations. A specific primary structure that directs folding to the pseudoknot or SLS is called SL-palindrome (SLP). Using a computer program for searching the SLP in the genomic sequences, 419 primary structures of large ribosomal RNAs from different kingdoms (prokaryota, eukaryota, archaebacteria) as well as plastids and mitochondria were analyzed. A universal site was found in the peptidyltransferase center (PTC) capable of folding to a pseudoknot of 48 nucleotides in length. Phylogenetic conservation of its helices (concurrent replacements with no violation of base pairing, covariation) has been demonstrated. We suggest the reversible folding-unfolding of the pseudoknot for certain stages of the ribosome functioning.

Database on the structure of large subunit ribosomal RNA.

The Antwerp database on large subunit ribosomal RNA now contains 607 complete or nearly complete aligned sequences. The alignment incorporates secondary structure information for each sequence. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available. Information from the database can be downloaded or searched on the rRNA WWW Server at URL http://rrna.uia.ac.be/

To Knot or Not to Knot? Examination of 16S Ribosomal RNA Models

The presence of topological knots in large RNA structures is highly unlikely given that 1) no RNA structures determined thus far contain topological knots, 2) secondary structure maps for most RNA molecules are knot free, 3) there are no known RNA topoisomerases, and 4) it is difficult to imagine how knots could be formed specifically and uniquely during transcription. Since native RNA structures probably lack topological knots, models of these RNA molecules should be free of knots as well. Therefore, we have examined four existing models for the 30S ribosomal subunit to determine if any of the three domains of the 16S rRNA molecule is knotted. We found that all but one model had at least one knotted domain. We conclude that models of large RNA molecules should be examined for knotting before publication.

MANIP: an interactive tool for modelling RNA

Large RNA structures can be viewed as assemblies of smaller units or modules that are usually clearly identified (helices, hairpin loops, other recurrent motifs, etc.). We have developed a program, MANIP, which allows the rapid assembly of separate motifs (each with a specified sequence) into a complex three-dimensional architecture. The already determined modules are present in a database from which they can be extracted with the appropriate sequence. Their assembly is performed in real time on the computer screen with buttons and dials that command rotation and translation of any chosen fragment with respect to the chosen pivot, or that generate all possible variations of any torsion angle within a specified segment either in the 5′ or in the 3′ direction. The possible in-built manipulations follow the general stereochemical rules of RNA structure. MANIP automatically recognizes and displays the allowed and nonallowed hydrogen bonds between the residues. The program is interfaced with a rapid and automatic online refinement tool of partial or full assemblies, NUCLIN-NUCLSQ. The refinement protocol incorporates canonical as well as noncanonical base pairing constraints together with restraints imposed by covalent geometry, stereochemistry, and van der Waals contacts. The computer package runs on UNIX Silicon Graphics workstations and is written in C with OpenGL and X11/Motif libraries.

MANIP: an interactive tool for modelling RNA

Large RNA structures can be viewed as assemblies of smaller units or modules that are usually clearly identified (helices, hairpin loops, other recurrent motifs, etc.). We have developed a program, MANIP, which allows the rapid assembly of separate motifs (each with a specified sequence) into a complex three-dimensional architecture. The already determined modules are present in a database from which they can be extracted with the appropriate sequence. Their assembly is performed in real time on the computer screen with buttons and dials that command rotation and translation of any chosen fragment with respect to the chosen pivot, or that generate all possible variations of any torsion angle within a specified segment either in the 5′ or in the 3′ direction. The possible in-built manipulations follow the general stereochemical rules of RNA structure. MANIP automatically recognizes and displays the allowed and nonallowed hydrogen bonds between the residues. The program is interfaced with a rapid and automatic online refinement tool of partial or full assemblies, NUCLIN-NUCLSQ. The refinement protocol incorporates canonical as well as noncanonical base pairing constraints together with restraints imposed by covalent geometry, stereochemistry, and van der Waals contacts. The computer package runs on UNIX Silicon Graphics workstations and is written in C with OpenGL and X11/Motif libraries.

Database on the structure of large ribosomal subunit RNA.

The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp.

Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site.

Helix packing is critical for RNA tertiary structure formation, although the rules for helix-helix association within structured RNAs are largely unknown. Docking of the substrate helix into the active site of the Tetrahymena group I ribozyme provides a model system to study this question. Using a novel chemogenetic method to analyze RNA structure in atomic detail, we report that complementary sets of noncanonical base pairs (a G.U wobble pair and two consecutively stacked sheared A.A pairs) create an RNA helix packing motif that is essential for 5'-splice site selection in the group I intron. This is likely to be a general motif for helix-helix interaction within the tertiary structures of many large RNAs.