Analysis of Four-Way Junctions in RNA Structures

RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.     

Promoting RNA helical stacking via A-minor junctions

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.     

Interconversion between Parallel and Antiparallel Conformations of a 4H RNA junction in Domain 3 of Foot-and-Mouth Disease Virus IRES Captured by Dynamics Simulations

RNA junctions are common secondary structural elements present in a wide range of RNA species. They play crucial roles in directing the overall folding of RNA molecules as well as in a variety of biological functions. In particular, there has been great interest in the dynamics of RNA junctions, including conformational pathways of fully base-paired 4-way (4H) RNA junctions. In such constructs, all nucleotides participate in one of the four double-stranded stem regions, with no connecting loops. Dynamical aspects of these 4H RNAs are interesting because frequent interchanges between parallel and antiparallel conformations are thought to occur without binding of other factors. Gel electrophoresis and single-molecule fluorescence resonance energy transfer experiments have suggested two possible pathways: one involves a helical rearrangement via disruption of coaxial stacking, and the other occurs by a rotation between the helical axes of coaxially stacked conformers. Employing molecular dynamics simulations, we explore this conformational variability in a 4H junction derived from domain 3 of the foot-and-mouth disease virus internal ribosome entry site (IRES); this junction contains highly conserved motifs for RNA-RNA and RNA-protein interactions, important for IRES activity. Our simulations capture transitions of the 4H junction between parallel and antiparallel conformations. The interconversion is virtually barrier-free and occurs via a rotation between the axes of coaxially stacked helices with a transient perpendicular intermediate. We characterize this transition, with various interhelical orientations, by pseudodihedral angle and interhelical distance measures. The high flexibility of the junction, as also demonstrated experimentally, is suitable for IRES activity. Because foot-and-mouth disease virus IRES structure depends on long-range interactions involving domain 3, the perpendicular intermediate, which maintains coaxial stacking of helices and thereby consensus primary and secondary structure information, may be beneficial for guiding the overall organization of the RNA system in domain 3.     

Predicting coaxial helical stacking in RNA junctions

RNA junctions are important structural elements that form when three or more helices come together in space in the tertiary structures of RNA molecules. Determining their structural configuration is important for predicting RNA 3D structure. We introduce a computational method to predict, at the secondary structure level, the coaxial helical stacking arrangement in junctions, as well as classify the junction topology. Our approach uses a data mining approach known as random forests, which relies on a set of decision trees trained using length, sequence and other variables specified for any given junction. The resulting protocol predicts coaxial stacking within three- and four-way junctions with an accuracy of 81% and 77%, respectively; the accuracy increases to 83% and 87%, respectively, when knowledge from the junction family type is included. Coaxial stacking predictions for the five to ten-way junctions are less accurate (60%) due to sparse data available for training. Additionally, our application predicts the junction family with an accuracy of 85% for three-way junctions and 74% for four-way junctions. Comparisons with other methods, as well applications to unsolved RNAs, are also presented. The web server Junction-Explorer to predict junction topologies is freely available at: http://bioinformatics.njit.edu/junction.     

Global structure of four-way RNA junctions studied using fluorescence resonance energy transfer.

Four-way helical junctions are found widely in natural RNA species. In this study, we have studied the conformation of two junctions by fluorescence resonance energy transfer. We show that the junctions are folded by pairwise coaxial helical stacking, forming one predominant stacking conformer in both examples studied. At low magnesium ion concentrations, the helical axes of both junctions are approximately perpendicular. One junction undergoes a rotation into a distorted antiparallel structure induced by the binding of a single magnesium ion. By contrast, the axes of the four-way junction of the U1 snRNA remain approximately perpendicular under all conditions examined, and we have determined the stacking conformer adopted.     

Annotation of tertiary interactions in RNA structures reveals variations and correlations

RNA tertiary motifs play an important role in RNA folding and biochemical functions. To help interpret the complex organization of RNA tertiary interactions, we comprehensively analyze a data set of 54 high-resolution RNA crystal structures for motif occurrence and correlations. Specifically, we search seven recognized categories of RNA tertiary motifs (coaxial helix, A-minor, ribose zipper, pseudoknot, kissing hairpin, tRNA D-loop/T-loop, and tetraloop-tetraloop receptor) by various computer programs. For the nonredundant RNA data set, we find 613 RNA tertiary interactions, most of which occur in the 16S and 23S rRNAs. An analysis of these motifs reveals the diversity and variety of A-minor motif interactions and the various possible loop-loop receptor interactions that expand upon the tetraloop-tetraloop receptor. Correlations between motifs, such as pseudoknot or coaxial helix with A-minor, reveal higher-order patterns. These findings may ultimately help define tertiary structure restraints for RNA tertiary structure prediction. A complete annotation of the RNA diagrams for our data set is available at http://www.biomath.nyu.edu/motifs/.     

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA''s nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single- or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction''s topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.     

Conformational flexibility of four-way junctions in RNA

Helical junctions are common architectural features in RNA. They are particularly important in autonomously folding molecules, as exemplified by the hairpin ribozyme. We have used single-molecule fluorescence spectroscopy to study the dynamic properties of the perfect (4H) four-way helical junction derived from the hairpin ribozyme. In the presence of Mg(2+), the junction samples parallel and antiparallel conformations and both stacking conformers, with a bias towards one antiparallel stacking conformer. There is continual interconversion between the forms, such that there are several transitions per second under physiological conditions. Our data suggest that interconversion proceeds via an open intermediate with reduced cation binding in which coaxial stacking between helices is disrupted. The rate of interconversion becomes slower at higher Mg(2+) concentrations, yet the activation barrier decreases under these conditions, indicating that entropic effects are important. Transitions also occur in the presence of Na(+) only; however, the coaxial stacking appears incomplete under these conditions. The polymorphic and dynamic character of the four-way RNA junction provides a source of structural diversity, from which particular conformations required for biological function might be stabilised by additional RNA interactions or protein binding.     

Helix Capping in RNA Structure

Helices are an essential element in defining the three-dimensional architecture of structured RNAs. While internal basepairs in a canonical helix stack on both sides, the ends of the helix stack on only one side and are exposed to the loop side, thus susceptible to fraying unless they are protected. While coaxial stacking has long been known to stabilize helix ends by directly stacking two canonical helices coaxially, based on analysis of helix-loop junctions in RNA crystal structures, herein we describe helix capping, topological stacking of a helix end with a basepair or an unpaired nucleotide from the loop side, which in turn protects helix ends. Beyond the topological protection of helix ends against fraying, helix capping should confer greater stability onto the resulting composite helices. Our analysis also reveals that this general motif is associated with the formation of tertiary structure interactions. Greater knowledge about the dynamics at the helix-junctions in the secondary structure should enhance the prediction of RNA secondary structure with a richer set of energetic rules and help better understand the folding of a secondary structure into its three-dimensional structure. These together suggest that helix capping likely play a fundamental role in driving RNA folding.     

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.     

New insights into the fundamental role of topological constraints as a determinant of two-way junction conformation

Recent studies have shown that topological constraints encoded at the RNA secondary structure level involving basic steric and stereochemical forces can significantly restrict the orientations sampled by helices across two-way RNA junctions. Here, we formulate these topological constraints in greater quantitative detail and use this topological framework to rationalize long-standing but poorly understood observations regarding the basic behavior of RNA two-way junctions. Notably, we show that the asymmetric nature of the A-form helix and the finite length of a bulge provide a physical basis for the experimentally observed directionality and bulge-length amplitude dependence of bulge induced inter-helical bends. We also find that the topologically allowed space can be modulated by variations in sequence, particularly with the addition of non-canonical GU base pairs at the junction, and, surprisingly, by the length of the 5' and 3' helices. A survey of two-way RNA junctions in the protein data bank confirms that junction residues have a strong preference to adopt looped-in, non-canonically base-paired conformations, providing a route for extending our bulge-directed framework to internal loop motifs and implying a simplified link between secondary and tertiary structure. Finally, our results uncover a new simple mechanism for coupling junction-induced topological constraints with tertiary interactions.     

Three-way RNA junctions with remote tertiary contacts: A recurrent and highly versatile fold

Three-way junction RNAs adopt a recurrent Y shape when two of the helices form a coaxial stack and the third helix establishes one or more tertiary contacts several base pairs away from the junction. In this review, the structure, distribution, and functional relevance of these motifs are examined. Structurally, the folds exhibit conserved junction topologies, and the distal tertiary interactions play a crucial role in determining the final shape of the structures. The junctions and remote tertiary contacts behave as flexible hinge motifs that respond to changes in the other region, providing these folds with switching mechanisms that have been shown to be functionally useful in a variety of contexts. In addition, the juxtaposition of RNA domains at the junction and at the distal tertiary complexes enables the RNA helices to adopt unusual conformations that are frequently used by proteins, RNA molecules, and antibiotics as platforms for specific binding. As a consequence of these properties, Y-shaped junctions are widely distributed in all kingdoms of life, having been observed in small naked RNAs such as riboswitches and ribozymes or embedded in complex ribonucleoprotein systems like ribosomal RNAs, RNase P, or the signal recognition particle. In all cases, the folds were found to play an essential role for the functioning or assembly of the RNA or ribonucleoprotein systems that contain them.     

Structure of the three-way helical junction of the hepatitis C virus IRES element

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS₄ junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS₁HS₃ and 2HS₂HS₂ junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg²⁺ ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.     

Computational investigation of the impact of core sequence on immobile DNA four-way junction structure and dynamics

Immobile four-way junctions (4WJs) are core structural motifs employed in the design of programmed DNA assemblies. Understanding the impact of sequence on their equilibrium structure and flexibility is important to informing the design of complex DNA architectures. While core junction sequence is known to impact the preferences for the two possible isomeric states that junctions reside in, previous investigations have not quantified these preferences based on molecular-level interactions. Here, we use all-atom molecular dynamics simulations to investigate base-pair level structure and dynamics of four-way junctions, using the canonical Seeman J1 junction as a reference. Comparison of J1 with equivalent single-crossover topologies and isolated nicked duplexes reveal conformational impact of the double-crossover motif. We additionally contrast J1 with a second junction core sequence termed J24, with equal thermodynamic preference for each isomeric configuration. Analyses of the base-pair degrees of freedom for each system, free energy calculations, and reduced-coordinate sampling of the 4WJ isomers reveal the significant impact base sequence has on local structure, isomer bias, and global junction dynamics.     

Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation

The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives internal initiation of viral protein synthesis during host cell infection. In the tertiary structure of the IRES RNA, two helical junctions create recognition sites for direct binding of the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The 2.8 A resolution structure of the IIIabc four-way junction, which is critical for binding eIF3, reveals how junction nucleotides interact with an adjacent helix to position regions directly involved in eIF3 recognition. Two of the emergent helices stack to form a nearly continuous A-form duplex, while stacking of the other two helices is interrupted by the insertion of junction residues into the helix minor groove. This distorted stack probably serves as an important recognition surface for the translational machinery.     

Predicting Helical Topologies in RNA Junctions as Tree Graphs

RNA molecules are important cellular components involved in many fundamental biological processes. Understanding the mechanisms behind their functions requires knowledge of their tertiary structures. Though computational RNA folding approaches exist, they often require manual manipulation and expert intuition; predicting global long-range tertiary contacts remains challenging. Here we develop a computational approach and associated program module (RNAJAG) to predict helical arrangements/topologies in RNA junctions. Our method has two components: junction topology prediction and graph modeling. First, junction topologies are determined by a data mining approach from a given secondary structure of the target RNAs; second, the predicted topology is used to construct a tree graph consistent with geometric preferences analyzed from solved RNAs. The predicted graphs, which model the helical arrangements of RNA junctions for a large set of 200 junctions using a cross validation procedure, yield fairly good representations compared to the helical configurations in native RNAs, and can be further used to develop all-atom models as we show for two examples. Because junctions are among the most complex structural elements in RNA, this work advances folding structure prediction methods of large RNAs. The RNAJAG module is available to academic users upon request.     

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.     

Modifications and deletions of helices within the hairpin ribozyme-substrate complex: an active ribozyme lacking helix 1

Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.     

Effect of single-residue bulges on RNA double-helical structures: crystallographic database analysis and molecular dynamics simulation studies

Asymmetric bulge loop motifs are widely dispersed in all types of functional RNAs. They are frequently occurring structural motifs in folded RNA structures and appear commonly in pre-microRNA and ribosomes, where they are involved in specific RNA–RNA and RNA–protein interactions. It is therefore necessary to understand such motifs from a structural point of view. We analyzed all available RNA structures and identified quite a few fragments of double helices that contain bulges. We found that these discontinuities often introduce kinks into the double helices, which also affects the stacking overlap between the base pairs across the irregularity. In order to understand the influence of these bulges on stability and flexibility, we carried out molecular dynamics simulations of three different single-residue bulge-containing RNA helices using the CHARMM36 force field. The structural variability at the junctions of RNA bulges is expected to differ from that in continuous double-helical stretches. The structural features of the junction region were observed to vary noticeably depending on the orientation of the bulge residue. When the base of the bulge residue is looped out, the RNA stretch behaves like a standard long A-form RNA double helix, whereas the entire RNA behaves differently when the base of the bulge residue is intercalated between base pairs inside the RNA stem. Such single-base intercalation was found to introduce a permanent kink into the composite double helix, which could be a recognition element for Dicer during the maturation of miRNA.