Elucidation of the structures of all members of the Avsunviroidae family

Viroids are small single‐stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and time‐consuming nature of classic approaches. Here, the method of high‐throughput selective 2′‐hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer‐assisted structure prediction using RNAstructure software in order to determine the secondary structures of the RNA strands of both (+) and (–) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (–) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid‐like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined. To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids.     

Compilation and analysis of viroid and viroid-like RNA sequences.

We have created a catalogue comprising all viroid and viroid-like RNA sequences which to our knowledge have been either published or were available from on-line sequence libraries as of October 1, 1995. In the development of this catalogue nomenclature ambiguities were removed, the likely ancestral sequence of most species was determined and the most stable secondary structures of these sequences were predicted using the MulFold package. Only viroids of PSTVd-type possessed a rod-like secondary structure, while most other viroids adopted branched secondary structures. Several viroids have predicted secondary structures that include either a Y or cruciform structure reminiscent of the tRNA-like end of virus genomes at an extremity. However, it remains unknown whether or not these predicted structures are adopted in solution, and if they serve a particular function in vivo. Additional information such as the position of the self-catalytic domains are included in the catalogue. An analysis of the data compilated in the catalogue is included. The catalogue will be available on the world wide web (http://www.callistro.si.usherb.ca/jpperra), on computer disk and in printed form. It should provide an excellent reference point for further studies.     

Developing three-dimensional models of putative-folding intermediates of the HDV ribozyme

Both the role and the interacting partners of an RNA molecule can change depending on its tertiary structure. Consequently, it is important to be able to accurately predict the complete folding pathway of an RNA molecule. The hepatitis delta virus (HDV) ribozyme is a small catalytic RNA with the greatest number of folding intermediates making it the model of choice with which to address this problem. The tertiary structures of the known putative intermediates along the folding pathway of the HDV ribozyme were predicted using the Macromolecular Conformations Symbolic programming (MC-Sym) software. The structures obtained by this method received physical support from Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). The analysis of these structures elucidated several features of the HDV ribozyme. In addition, this report represents an application for MC-Sym that permits progression one step further toward the computer prediction of an RNA molecule-folding pathway.     

Characteristic chemical probing patterns of loop motifs improve prediction accuracy of RNA secondary structures

RNA structures play a fundamental role in nearly every aspect of cellular physiology and pathology. Gaining insights into the functions of RNA molecules requires accurate predictions of RNA secondary structures. However, the existing thermodynamic folding models remain less accurate than desired, even when chemical probing data, such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reactivities, are used as restraints. Unlike most SHAPE-directed algorithms that only consider SHAPE restraints for base pairing, we extract two-dimensional structural features encoded in SHAPE data and establish robust relationships between characteristic SHAPE patterns and loop motifs of various types (hairpin, internal, and bulge) and lengths (2–11 nucleotides). Such characteristic SHAPE patterns are closely related to the sugar pucker conformations of loop residues. Based on these patterns, we propose a computational method, SHAPELoop, which refines the predicted results of the existing methods, thereby further improving their prediction accuracy. In addition, SHAPELoop can provide information about local or global structural rearrangements (including pseudoknots) and help researchers to easily test their hypothesized secondary structures.     

Correlating SHAPE signatures with three-dimensional RNA structures

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a facile technique for quantitative analysis of RNA secondary structure. In general, low SHAPE signal values indicate Watson-Crick base-pairing, and high values indicate positions that are single-stranded within the RNA structure. However, the relationship of SHAPE signals to structural properties such as non-Watson-Crick base-pairing or stacking has thus far not been thoroughly investigated. Here, we present results of SHAPE experiments performed on several RNAs with published three-dimensional structures. This strategy allows us to analyze the results in terms of correlations between chemical reactivities and structural properties of the respective nucleotide, such as different types of base-pairing, stacking, and phosphate-backbone interactions. We find that the RNA SHAPE signal is strongly correlated with cis-Watson-Crick/Watson-Crick base-pairing and is to a remarkable degree not dependent on other structural properties with the exception of stacking. We subsequently generated probabilistic models that estimate the likelihood that a residue with a given SHAPE score participates in base-pairing. We show that several models that take SHAPE scores of adjacent residues into account perform better in predicting base-pairing compared with individual SHAPE scores. This underscores the context sensitivity of SHAPE and provides a framework for an improved interpretation of the response of RNA to chemical modification.     

Comparative sequence and structure of viroid-like RNAs of two plant viruses.

A newly discovered group of spherical plant viruses contains a bipartite genome consisting of a single-strand linear RNA molecule (RNA 1, Mr 1.5 x 10(6) ), and a single-strand, covalently closed circular viroid-like RNA molecule (RNA 2, Mr approximately 125,000). The nucleotide sequences of the RNA 2 of two of these, velvet tobacco mottle virus and solanum nodiflorum mottle virus, have been determined. RNA 2 of solanum nodiflorum mottle virus consists of 377 residues whereas that of velvet tobacco mottle virus consists of two approximately equimolar species, one of 366 residues and the other, with a single nucleotide deletion, of 365 residues. There is 92-95% sequence homology between the RNA 2 species of the two viruses. The predicted secondary structures possess extensive intramolecular base pairing to give rod-like structures similar to those of viroids. The structural similarities between the RNAs 2 of velvet tobacco mottle virus and solanum nodiflorum mottle virus and viroids may reflect functional similarities.     

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.     

Dynamics and interactions of viroids

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.     

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.     

Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.     

R-CHIE: a web server and R package for visualizing RNA secondary structures

Visually examining RNA structures can greatly aid in understanding their potential functional roles and in evaluating the performance of structure prediction algorithms. As many functional roles of RNA structures can already be studied given the secondary structure of the RNA, various methods have been devised for visualizing RNA secondary structures. Most of these methods depict a given RNA secondary structure as a planar graph consisting of base-paired stems interconnected by roundish loops. In this article, we present an alternative method of depicting RNA secondary structure as arc diagrams. This is well suited for structures that are difficult or impossible to represent as planar stem-loop diagrams. Arc diagrams can intuitively display pseudo-knotted structures, as well as transient and alternative structural features. In addition, they facilitate the comparison of known and predicted RNA secondary structures. An added benefit is that structure information can be displayed in conjunction with a corresponding multiple sequence alignments, thereby highlighting structure and primary sequence conservation and variation. We have implemented the visualization algorithm as a web server R-chie as well as a corresponding R package called R4RNA, which allows users to run the software locally and across a range of common operating systems.     

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.     

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5–3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson–Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.     

Dynamics and Interactions of Viroids

Abstract

Viroids are single stranded circular RNA molecules of 120 000 dal tons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids.

For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure.

The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.     

Formation of spatial structure of RNA molecules

In this review we consider several experimental and theoretical approaches for investigation of RNA folding and determination of nucleotides that play an important role upon folding of such molecules as tRNA and several classes of ribozymes. It has been shown that nucleotides in the D- and T-loop regions are the last to be involved in tRNA structure or they are not included in the folding nucleus of tRNA. Using the specially elaborated method SHAPE it has been demonstrated that the model of hierarchical folding which was recognized for a long time is not correct for tRNA folding. In the second part of the given review the algorithms and programs used for the prediction of secondary structures of RNA as well as for modeling of RNA folding are considered.     

Common structural features of different viroids: serial arrangement of double helical sections and internal loops.

The thermodynamic parameters of five different highly purified viroid "species" were determined by applying UV-absorption melting analysis and temperature jump methods. Their thermal denaturation proved to be a highly cooperative process with midpoint-temperatures (Tm) between 48.5 and 51 degrees C in 0.01 M sodium cacodylate, 1 mM EDTA, pH 6.8. The values of the apparent reaction enthalpies of the different viroid species range between 3,140 and 3,770 kJ/mol. Although the cooperativity is as high as found in homogeneous RNA double helices the Tm-value of viroid melting is more than 30 degrees C lower than in the homogeneous RNA. In order to explain this deviation, melting curves were simulated for different models of the secondary structure of viroids using literature values of the thermodynamic parameters of nucleic acids. Our calculations show that the following refinement of our earlier model is in complete accordance with the experimental data: In their native conformation viroids exist as an extended rodlike structure characterized by a series of double helical sections and internal loops. In the different viroid species 250-300 nucleotides out of total 350 nucleotides are needed to interprete the thermodynamic behaviour.