RNA Three-dimensional Structures Research Articles

RNA structure: new messages in translation, replication and disease

The ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs took place between 27 and 29 October 2008, in Baeza, Spain, and was organized by E. Martinez‐Salas, G. Belsham & J. Gomez. ![][1] See Glossary for abbreviations used in this article Since the elucidation of the genetic code approximately 40 years ago, the role of RNA in crucial biological processes has gained increasing attention, and is now one of the fastest developing areas in molecular biology and biochemistry. The advances made in structural studies of transfer RNA (tRNA), ribozymes, riboswitches, the ribosome and viral IRES elements in particular have shown the importance of secondary and tertiary structures in RNA biology, and have significantly changed our understanding of the translational control of gene expression (Fig 1). The World Heritage town of Baeza in Andalucia, Spain, hosted the ‘Current Trends in Biomedicine’ workshop on the role of RNA structures in the translation of viral and cellular RNAs. About 45 participants met in the beautiful buildings of the Sede Antonio Machado at the Universidad Internacional de Andalucia. Figure 1. Schematic illustration of discreet RNA regulatory elements. The translation of an mRNA is regulated by diverse mechanisms that involve both structural and non‐structural RNA elements, as well as interactions with RNA‐binding proteins. IRESes are found in the 5′ UTR and promote cap‐independent translation. Pseudoknots can be located in the 5′ UTR, the 3′ UTR, or the coding region, and their localization influences their effect on translation; for example, initiation, frameshifting and termination. 3′ UTR structural elements such as CREs, CITEs and SLs often function through long‐range RNA interactions. The miRNA target sites are located in the 3′ UTR. The canonical mRNA features of the cap (m7G) and poly(A) are targets of several regulatory interactions. The … [1]: /embed/graphic-1.gif

RNA structure alignment by a unit-vector approach

The recent discovery of tiny RNA molecules such as microRNAs and small interfering RNA are transforming the view of RNA as a simple information transfer molecule. Similar to proteins, the native three-dimensional structure of RNA determines its biological activity. Therefore, classifying the current structural space is paramount for functionally annotating RNA molecules. The increasing numbers of RNA structures deposited in the PDB requires more accurate, automatic and benchmarked methods for RNA structure comparison. In this article, we introduce a new algorithm for RNA structure alignment based on a unit-vector approach. The algorithm has been implemented in the SARA program, which results in RNA structure pairwise alignments and their statistical significance. The SARA program has been implemented to be of general applicability even when no secondary structure can be calculated from the RNA structures. A benchmark against the ARTS program using a set of 1275 non-redundant pairwise structure alignments results in inverted approximately 6% extra alignments with at least 50% structurally superposed nucleotides and base pairs. A first attempt to perform RNA automatic functional annotation based on structure alignments indicates that SARA can correctly assign the deepest SCOR classification to >60% of the query structures. The SARA program is freely available through a World Wide Web server http://sgu.bioinfo.cipf.es/services/SARA/. Supplementary data are available at Bioinformatics online.

IFoldRNA: three-dimensional RNA structure prediction and folding

Three-dimensional RNA structure prediction and folding is of significant interest in the biological research community. Here, we present iFoldRNA, a novel web-based methodology for RNA structure prediction with near atomic resolution accuracy and analysis of RNA folding thermodynamics. iFoldRNA rapidly explores RNA conformations using discrete molecular dynamics simulations of input RNA sequences. Starting from simplified linear-chain conformations, RNA molecules (<50 nt) fold to native-like structures within half an hour of simulation, facilitating rapid RNA structure prediction. All-atom reconstruction of energetically stable conformations generates iFoldRNA predicted RNA structures. The predicted RNA structures are within 2-5 A root mean squre deviations (RMSDs) from corresponding experimentally derived structures. RNA folding parameters including specific heat, contact maps, simulation trajectories, gyration radii, RMSDs from native state, fraction of native-like contacts are accessible from iFoldRNA. We expect iFoldRNA will serve as a useful resource for RNA structure prediction and folding thermodynamic analyses. http://iFoldRNA.dokhlab.org.

Structural Principles From Large RNAs

Since the year 2000 a number of large RNA three-dimensional structures have been determined by X-ray crystallography. Structures composed of more than 100 nucleotide residues include the signal recognition particle RNA, group I intron, the GlmS ribozyme, RNAseP RNA, and ribosomal RNAs from Haloarcula morismortui, Escherichia coli, Thermus thermophilus, and Deinococcus radiodurans. These large RNAs are constructed from the same secondary and tertiary structural motifs identified in smaller RNAs but appear to have a larger organizational architecture. They are dominated by long continuous interhelical base stacking, tend to segregate into domains, and are planar in overall shape as opposed to their globular protein counterparts. These findings have consequences in RNA folding, intermolecular interaction, and packing, in addition to studies of design and engineering and structure prediction.

ALEXANDER RICH WINS WELCH AWARD

FOR HIS PIONEERING WORK on nucleic acids, Alexander Rich, a professor of biophysics at MIT, has won the 2008 Welch Award in Chemistry. The $300,000 award is given annually by Houston’s Welch Foundation to foster and encourage basic chemical research that benefits humankind. A student of Linus Pauling’s and a contemporary of James Watson and Francis Crick’s, Rich has devoted his entire career to studying DNA and RNA. In 1979, he and his colleagues discovered left-handed DNA, which they named Z-DNA for its zigzag backbone. Since then, Rich’s research has focused on Z-DNA’s importance in biological systems. In addition, Rich discovered and solved the three-dimensional structure of the RNA double helix. He was the first scientist to carry out DNA-RNA hybridization and to discover DNA’s presence in organelles. He also discovered polyribosomes, which are clusters of ribosomes attached to messenger RNA, and determined the three-dimensional structure of transfer RNA. Rich is known for his enthusiasm ...

Ab initio RNA folding by discrete molecular dynamics: From structure prediction to folding mechanisms

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 A deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA(Phe), pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.

Structure, Stability, and Dynamics of Canonical and Noncanonical Base Pairs: Quantum Chemical Studies

The importance of non-Watson-Crick base pairs in the three-dimensional structure of RNA is now well established. The structure and stability of these noncanonical base pairs are, however, poorly understood. We have attempted to understand structural features of 33 frequently occurring base pairs using density functional theory. These are of three types, namely (i) those stabilized by two or more polar hydrogen bonds between the bases, (ii) those having one polar and another C-H...O/N type interactions, and (iii) those having one H-bond between the bases and another involving one of the sugars linked to the bases. We found that the base pairs having two polar H-bonds are very stable as compared to those having one C-H...O/N interaction. Our quantitatively analysis of structures of these optimized base pairs indicates that they possess a different amount of nonplanarity with large propeller or buckle values as also observed in the crystal structures. We further found that geometry optimization does not modify the hydrogen-bonding pattern, as values of shear and open angle of the base pairs remain conserved. The structures of initial crystal geometry and final optimized geometry of some base pairs having only one polar H-bond and a C-H...O/N interaction, however, are significantly different, indicating the weak nature of the nonpolar interaction. The base pair flexibility, as measured from normal-mode analysis, in terms of the intrinsic standard deviations of the base pair structural parameters are in conformity with those calculated from RNA crystal structures. We also noticed that deformation of a base pair along the stretch direction is impossible for all of the base pairs, and movements of the base pairs along shear and open are also quite restricted. The base pair opening mode through alteration of propeller or buckle is considerably less restricted for most of the base pairs.

The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data

The classical RNA secondary structure model considers A.U and G.C Watson-Crick as well as G.U wobble base pairs. Here we substitute it for a new one, in which sets of nucleotide cyclic motifs define RNA structures. This model allows us to unify all base pairing energetic contributions in an effective scoring function to tackle the problem of RNA folding. We show how pipelining two computer algorithms based on nucleotide cyclic motifs, MC-Fold and MC-Sym, reproduces a series of experimentally determined RNA three-dimensional structures from the sequence. This demonstrates how crucial the consideration of all base-pairing interactions is in filling the gap between sequence and structure. We use the pipeline to define rules of precursor microRNA folding in double helices, despite the presence of a number of presumed mismatches and bulges, and to propose a new model of the human immunodeficiency virus-1 -1 frame-shifting element.

IFoldRNA: three-dimensional RNA structure prediction and folding

iFoldRNA: three-dimensional RNA structure prediction and folding

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen.

The life of aerobes is dependent on iron and oxygen for efficient bioenergetics. Due to potential risks associated with iron/oxygen chemistry, iron acquisition, concentration, storage, utilization, and efflux are tightly regulated in the cell. A central role in regulating iron/oxygen chemistry in animals is played by mRNA translation or turnover via the iron responsive element (IRE)/iron regulatory protein (IRP) system. The IRE family is composed of three-dimensional RNA structures located in 3' or 5' untranslated regions of mRNA. To date, there are 11 different IRE mRNAs in the family, regulated through translation initiation or mRNA stability. Iron or oxidant stimuli induce a set of graded responses related to mRNA-specific IRE substructures, indicated by differential responses to iron in vivo and binding IRPs in vitro. Molecular effects of phosphorylation, iron and oxygen remain to be added to the structural information of the IRE-RNA and IRP repressor in the regulatory complex.

The Three-dimensional Structure of Genomic RNA in Bacteriophage MS2: Implications for Assembly

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density encompassing ∼90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.

DIAL: a web server for the pairwise alignment of two RNA three-dimensional structures using nucleotide, dihedral angle and base-pairing similarities

DIAL (dihedral alignment) is a web server that provides public access to a new dynamic programming algorithm for pairwise 3D structural alignment of RNA. DIAL achieves quadratic time by performing an alignment that accounts for (i) pseudo-dihedral and/or dihedral angle similarity, (ii) nucleotide sequence similarity and (iii) nucleotide base-pairing similarity.DIAL provides access to three alignment algorithms: global (Needleman–Wunsch), local (Smith–Waterman) and semiglobal (modified to yield motif search). Suboptimal alignments are optionally returned, and also Boltzmann pair probabilities Pr(ai,bj) for aligned positions ai , bj from the optimal alignment. If a non-zero suboptimal alignment score ratio is entered, then the semiglobal alignment algorithm may be used to detect structurally similar occurrences of a user-specified 3D motif. The query motif may be contiguous in the linear chain or fragmented in a number of noncontiguous regions.The DIAL web server provides graphical output which allows the user to view, rotate and enlarge the 3D superposition for the optimal (and suboptimal) alignment of query to target. Although graphical output is available for all three algorithms, the semiglobal motif search may be of most interest in attempts to identify RNA motifs. DIAL is available at http://bioinformatics.bc.edu/clotelab/DIAL.

Stacking Effects on Local Structure in RNA: Changes in the Structure of Tandem GA Pairs when Flanking GC Pairs Are Replaced by isoG−isoC Pairs

The Watson-Crick-like isoG-isoC (iGiC) pair, with the amino and carbonyl groups transposed relative to the Watson-Crick GC pair, provides an expanded alphabet for understanding interactions that shape nucleic acid structure. Here, thermodynamic stabilities of tandem GA pairs flanked by iGiC pairs are reported along with the NMR structures of the RNA self-complementary duplexes (GCiGGAiCGCA)2 and (GGiCGAiGCCA)2. A sheared GA pairing forms in (GCiGGAiCGCA)2, and an imino GA pairing forms in (GGiCGAiGCCA)2. The structures contrast with the formation of tandem imino and sheared GA pairs flanked by GC pairs in the RNA self-complementary duplexes (GCGGACGC)2 and (GGCGAGCC)2, respectively. In both iGiC duplexes, Watson-Crick-like hydrogen bonds are formed between iG and iC, and iGiC substitutions result in less favorable loop stability. The results provide benchmarks for testing computations of molecular interactions that shape RNA three-dimensional structure.

Coplanar and coaxial orientations of RNA bases and helices

Electrostatic interactions, base-pairing, and especially base-stacking dominate RNA three-dimensional structures. In an A-form RNA helix, base-stacking results in nearly perfect parallel orientations of all bases in the helix. Interestingly, when an RNA structure containing multiple helices is visualized at the atomic level, it is often possible to find an orientation such that only the edges of most bases are visible. This suggests that a general aspect of higher level RNA structure is a coplanar arrangement of base-normal vectors. We have analyzed all solved RNA crystal structures to determine the degree to which RNA base-normal vectors are globally coplanar. Using a statistical test based on the Watson-Girdle distribution, we determined that 330 out of 331 known RNA structures show statistically significant (p < 0.05; false discovery rate [FDR] = 0.05) coplanar normal vector orientations. Not surprisingly, 94% of the helices in RNA show bipolar arrangements of their base-normal vectors (p < 0.05). This allows us to compute a mean axis for each helix and compare their orientations within an RNA structure. This analysis revealed that 62% (208/331) of the RNA structures exhibit statistically significant coaxial packing of helices (p < 0.05, FDR = 0.08). Further analysis reveals that the bases in hairpin loops and junctions are also generally planar. This work demonstrates coplanar base orientation and coaxial helix packing as an emergent behavior of RNA structure and may be useful as a structural modeling constraint.

High-throughput method for the prediction of low-resolution, three-dimensional RNA structures

Our aim is the high-throughput prediction of three-dimensional RNA structures at low resolution to answer questions posed by the RNA molecular biology. The strategy presented here is based on a computer program, called 3D-RNApredict, which implements and converts various structural data (RNA secondary structure, RNA motifs coordinates, experimental data) to create the input to the CYANA program (torsion-angle dynamics algorithm, TAD) which provides a fast engine for the 3D RNA structure calculation. The resultant RNA structure is refined using the X-PLOR program. The approach is amenable to automatization. Current predictions involve HIV-2 leader RNA domains and the sets of bacterial ncRNA.

Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package

BackgroundAvailability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA) for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment.ResultsThree-dimensional structure of rRNA is visualized in OpenGL 3D environment with the abilities to change the display and overlay information onto the molecule, dynamically. Phylogenetic information derived from the multiple sequence alignments can be overlaid onto the molecule structure in a real time. Superimposition of both statistical and non-statistical sequence associated information onto the rRNA 3D structure can be done using customizable color scheme, which is also applied to a textual sequence alignment for reference. Oligonucleotide probes designed by ARB probe design tools can be mapped onto the 3D structure along with the probe accessibility models for evaluation with respect to secondary and tertiary structural conformations of rRNA.ConclusionVisualization of three-dimensional structure of rRNA in an intuitive display provides the biologists with the greater possibilities to carry out structure based phylogenetic analysis. Coupled with secondary structure models of rRNA, RNA3D program aids in validating the sequence alignments of rRNA genes and evaluating probe target sites. Superimposition of the information derived from the multiple sequence alignment onto the molecule dynamically allows the researchers to observe any sequence inherited characteristics (phylogenetic information) in real-time environment. The extended ARB software package is made freely available for the scientific community via .

The RNA Ontology Consortium: An open invitation to the RNA community

The aim of the RNA Ontology Consortium (ROC) is to create an integrated conceptual framework-an RNA Ontology (RO)-with a common, dynamic, controlled, and structured vocabulary to describe and characterize RNA sequences, secondary structures, three-dimensional structures, and dynamics pertaining to RNA function. The RO should produce tools for clear communication about RNA structure and function for multiple uses, including the integration of RNA electronic resources into the Semantic Web. These tools should allow the accurate description in computer-interpretable form of the coupling between RNA architecture, function, and evolution. The purposes for creating the RO are, therefore, (1) to integrate sequence and structural databases; (2) to allow different computational tools to interoperate; (3) to create powerful software tools that bring advanced computational methods to the bench scientist; and (4) to facilitate precise searches for all relevant information pertaining to RNA. For example, one initial objective of the ROC is to define, identify, and classify RNA structural motifs described in the literature or appearing in databases and to agree on a computer-interpretable definition for each of these motifs. To achieve these aims, the ROC will foster communication and promote collaboration among RNA scientists by coordinating frequent face-to-face workshops to discuss, debate, and resolve difficult conceptual issues. These meeting opportunities will create new directions at various levels of RNA research. The ROC will work closely with the PDB/NDB structural databases and the Gene, Sequence, and Open Biomedical Ontology Consortia to integrate the RO with existing biological ontologies to extend existing content while maintaining interoperability.

Predicting RNA folding thermodynamics with a reduced chain representation model

Based on the virtual bond representation for the nucleotide backbone, we develop a reduced conformational model for RNA. We use the experimentally measured atomic coordinates to model the helices and use the self-avoiding walks in a diamond lattice to model the loop conformations. The atomic coordinates of the helices and the lattice representation for the loops are matched at the loop-helix junction, where steric viability is accounted for. Unlike the previous simplified lattice-based models, the present virtual bond model can account for the atomic details of realistic three-dimensional RNA structures. Based on the model, we develop a statistical mechanical theory for RNA folding energy landscapes and folding thermodynamics. Tests against experiments show that the theory can give much more improved predictions for the native structures, the thermal denaturation curves, and the equilibrium folding/unfolding pathways than the previous models. The application of the model to the P5abc region of Tetrahymena group I ribozyme reveals the misfolded intermediates as well as the native-like intermediates in the equilibrium folding process. Moreover, based on the free energy landscape analysis for each and every loop mutation, the model predicts five lethal mutations that can completely alter the free energy landscape and the folding stability of the molecule.

Extracting Stacking Interaction Parameters for RNA from the Data Set of Native Structures

A crucial step in the determination of the three-dimensional native structures of RNA is the prediction of their secondary structures, which are stable independent of the tertiary fold. Accurate prediction of the secondary structure requires context-dependent estimates of the interaction parameters. We have exploited the growing database of natively folded RNA structures in the Protein Data Bank (PDB) to obtain stacking interaction parameters using a knowledge-based approach. Remarkably, the calculated values of the resulting statistical potentials (SPs) are in excellent agreement with the parameters determined using measurements in small oligonucleotides. We validate the SPs by predicting 74% of the base-pairs in a dataset of structures using the ViennaRNA package. Interestingly, this number is similar to that obtained using the measured thermodynamic parameters. We also tested the efficacy of the SP in predicting secondary structure by using gapless threading, which we advocate as an alternative method for rapidly predicting RNA structures. For RNA molecules with less than 700 nucleotides, about 70% of the native base-pairs are correctly predicted. As a further validation of the SPs we calculated Z-scores, which measure the relative stability of the native state with respect to a manifold of higher free energy states. The computed Z-scores agree with estimates made using calorimetric measurements for a few RNA molecules. Structural analysis was used to rationalize the success and failures of SP and experimentally determined parameters. First, from the near perfect linear relationship between the number of native base-pairs and sequence length, we show that nearly 46% of nucleotides are not in stacks. Second, by analyzing the suboptimal structures that are generated in gapless threading we show that the SPs and experimentally determined parameters are most successful in predicting stacks that end in hairpins. These results show that further improvement in secondary structure prediction requires reliable estimates of interaction parameters for loops, bulges, and stacks that do not end in hairpins.

Toward multiplexing the application of solvent accessibility probes for the investigation of RNA three-dimensional structures by electrospray ionization–Fourier transform mass spectrometry

Multiple solvent accessibility probes can be applied simultaneously to investigate the three-dimensional structure of complex RNA substrates when electrospray ionization–Fourier transform mass spectrometry (ESI–FTMS) is employed in place of polyacrylamide gel electrophoresis (PAGE). We show that classic chemical probes, such as dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho- p-toluenesulfonate, can be combined in probing mixtures designed to assess the full spectrum of base pairing and steric protection for the most abundant ribonucleotides included in RNA. After probe-independent hydrolysis of the alkylated substrate, the mixture of oligonucleotide products is mass mapped by ESI–FTMS analysis, which enables the unambiguous identification of probed bases from the unique mass signatures provided by the different chemical modifiers. In this bottom-up approach, any theoretical limit to the size of the possible target RNA will be determined by the effectiveness of the hydrolysis procedure rather than by the performance of the detection technique. Control experiments performed on the stem–loop 4 of human immunodeficiency virus type 1 have shown no adverse interactions between the reagents combined in the probing cocktails. No significant discrepancies between the alkylation patterns offered by the cocktails and the individual reagents could be detected, indicating that multiplexing the probe application does not necessarily lead to structural distortion but provides valid data on base accessibility and protection. To demonstrate the ruggedness of this approach, optimized cocktails were finally employed to assess the stability of the folded structure of mouse mammary tumor virus pseudoknot in the presence of different amounts of Mg 2+. Multiplexing the probe application constitutes an essential step toward high-throughput applications, which will take advantage of a strategy that maximizes the information attainable from a single experiment, while minimizing time and sample consumption over PAGE-based methods.