RNA Tertiary Structure Research Articles

Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.

A new way to see RNA

Unlike proteins, the RNA backbone has numerous degrees of freedom (eight, if one counts the sugar pucker), making RNA modeling, structure building and prediction a multidimensional problem of exceptionally high complexity. And yet RNA tertiary structures are not infinite in their structural morphology; rather, they are built from a limited set of discrete units. In order to reduce the dimensionality of the RNA backbone in a physically reasonable way, a shorthand notation was created that reduced the RNA backbone torsion angles to two (η and θ, analogous to φ and ψ in proteins). When these torsion angles are calculated for nucleotides in a crystallographic database and plotted against one another, one obtains a plot analogous to a Ramachandran plot (the η/θ plot), with highly populated and unpopulated regions. Nucleotides that occupy proximal positions on the plot have identical structures and are found in the same units of tertiary structure. In this review, we describe the statistical validation of the η/θ formalism and the exploration of features within the η/θ plot. We also describe the application of the η/θ formalism in RNA motif discovery, structural comparison, RNA structure building and tertiary structure prediction. More than a tool, however, the η/θ formalism has provided new insights into RNA structure itself, revealing its fundamental components and the factors underlying RNA architectural form.

Automated RNA tertiary structure prediction from secondary structure and low-resolution restraints.

A novel protocol for all-atom RNA tertiary structure prediction is presented that uses restrained molecular mechanics and simulated annealing. The restraints are from secondary structure, covariation analysis, coaxial stacking predictions for helices in junctions, and, when available, cross-linking data. Results are demonstrated on the Alu domain of the mammalian signal recognition particle RNA, the Saccharomyces cerevisiae phenylalanine tRNA, the hammerhead ribozyme, the hepatitis C virus internal ribosomal entry site, and the P4-P6 domain of the Tetrahymena thermophila group I intron. The predicted structure is selected from a pool of decoy structures with a score that maximizes radius of gyration and base-base contacts, which was empirically found to select higher quality decoys. This simple ab initio approach is sufficient to make good predictions of the structure of RNAs compared to current crystal structures using both root mean square deviation and the accuracy of base-base contacts.

Improvements of the Hierarchical Approach for Predicting RNA Tertiary Structure

Computational prediction of RNA tertiary structures is a significant challenge, especially for longer RNA and pseudoknots. At present it is still difficult to do this by pure all-atom molecular dynamics simulation. One of possible approaches is through hierarchical steps: from sequence to secondary structure and then to tertiary structure. Here we present improvements of two key steps of this approach, the manual adjustment of atom clashes and bond stretches and molecular dynamics refinement. We provide an energy function to find the locations of atom clashes and bond stretches and to guide their manual adjustment and a new scheme of molecular dynamics refinement using a tested combination of solvent model and the ff98 Amber force field suitable for RNA. We predicted with higher accuracy the tertiary structures of nine typical RNA molecules of lengths from 12 to 52, including hairpins, duplex helices and pseudoknots.

Single-Molecule FRET Studies of Counterion Effects on the Free Energy Landscape of Human Mitochondrial Lysine tRNA

The folding energy landscape of RNA is greatly affected by interactions between the RNA and counterions that neutralize the backbone negative charges and may also participate in tertiary contacts. Valence, size, coordination number, and electron shell structure can all contribute to the energetic stabilization of specific RNA conformations. Using single-molecule fluorescence resonance energy transfer (smFRET), we have examined the folding properties of the RNA transcript of human mitochondrial tRNA(Lys), which possesses two different folded states in addition to the unfolded one under conditions of thermodynamic equilibrium. We have quantitatively analyzed the degree of RNA tertiary structure stabilization for different types of cations based on a thermodynamic model that accounts for multiple conformational states and RNA-ion interactions within each state. We have observed that small monovalent ions stabilize the tRNA tertiary structure more efficiently than larger ones. More ions were found in close vicinity of compact RNA structures, independent of the type of ion. The largest conformation-dependent binding specificity of ions of the same charge was found for divalent ions, for which the ionic radii and coordination properties were responsible for shaping the folding free energy.

A structural determinant required for RNA editing

RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing.

Effects of Mg2+ on the Free Energy Landscape for Folding a Purine Riboswitch RNA

There are potentially several ways Mg2+ might promote formation of an RNA tertiary structure: by causing a general "collapse" of the unfolded ensemble to more compact conformations, by favoring a reorganization of structure within a domain to a form with specific tertiary contacts, and by enhancing cooperative linkages between different sets of tertiary contacts. To distinguish these different modes of action, we have studied Mg2+ interactions with the adenine riboswitch, in which a set of tertiary interactions that forms around a purine-binding pocket is thermodynamically linked to the tertiary "docking" of two hairpin loops in another part of the molecule. Each of four RNA forms with different extents of tertiary structure were characterized by small-angle X-ray scattering. The free energy of interconversion between different conformations in the absence of Mg2+ and the free energy of Mg2+ interaction with each form have been estimated, yielding a complete picture of the folding energy landscape as a function of Mg2+ concentration. At 1 mM Mg2+ (50 mM K+), the overall free energy of stabilization by Mg2+ is large, -9.8 kcal/mol, and about equally divided between its effect on RNA collapse to a partially folded structure and on organization of the binding pocket. A strong cooperative linkage between the two sets of tertiary contacts is intrinsic to the RNA. This quantitation of the effects of Mg2+ on an RNA with two distinct sets of tertiary interactions suggests ways that Mg2+ may work to stabilize larger and more complex RNA structures.

The Role of Electrostatic Relaxation on the Folding Kinetics of a Bacterial Ribozyme

The self-assembly of catalytic RNA ribozymes into compact, native structures is critical for their functions in the cell. The first step in forming RNA tertiary structure is the neutralization by cations of the negative charges of the phosphates. This electrostatic stabilization enables dynamical exploration of more compact conformations, and the search for long-range tertiary interactions. Our previous time-resolved Small Angle X-ray Scattering (SAXS) studies showed that the Azoarcus ribozyme exhibits triphasic folding kinetics: up to 90% folds in less than 10 ms, which we attribute to specific collapse.

Precise Mapping of RNA Tertiary Structure via Nanometer Distance Measurements with Double Electron-Electron Resonance (DEER) Spectroscopy

Divalent metal (Mg(2+)) ion-dependent folding of the hammerhead ribozyme from Schistosoma mansoni was monitored with double electron-electron resonance (DEER) pulsed electron paramagnetic resonance spectroscopy by measuring nanometer-scale distances between paramagnetic spin-labels attached to the RNA. DEER measurements detect global folding of the ribozyme with excellent agreement between predictions from experimental, modeled, and crystallographic measurements. These measurements demonstrate the use of DEER spectroscopy as a tool for structural analysis of complex RNAs.

The RNA Ontology (RNAO): An ontology for integrating RNA sequence and structure data

Biomedical Ontologies integrate diverse biomedical data and enable intelligent data-mining and help translate basic research into useful clinical knowledge. We present the RNA Ontology (RNAO), an ontology for integrating diverse RNA data, including RNA sequences and sequence alignments, three-dimensional structures, and biochemical and functional data. For example, individual atomic resolution RNA structures have broader significance as representatives of classes of homologous molecules, which can differ significantly in sequence while sharing core structural features and common roles or functions. Thus, structural data gain value by being linked to homologous sequences in genomic data and databases of sequence alignments. Likewise, the value of genomic data is enhanced by annotation of shared structural features, especially when these can be linked to specific functions. Moreover, the significance of biochemical, functional and mutational analyses of RNA molecules are most fully understood when linked to molecular structures and phylogenies. To achieve these goals, RNAO provides logically rigorous definitions of the components of RNA primary, secondary and tertiary structure and the relations between these entities. RNAO is being developed to comply with the developing standards of the Open Biomedical Ontologies (OBO) Consortium. The RNAO can be accessed at http://code.google.com/p/rnao/.

Heuristic RNA pseudoknot prediction including intramolecular kissing hairpins

Pseudoknots are an essential feature of RNA tertiary structures. Simple H-type pseudoknots have been studied extensively in terms of biological functions, computational prediction, and energy models. Intramolecular kissing hairpins are a more complex and biologically important type of pseudoknot in which two hairpin loops form base pairs. They are hard to predict using free energy minimization due to high computational requirements. Heuristic methods that allow arbitrary pseudoknots strongly depend on the quality of energy parameters, which are not yet available for complex pseudoknots. We present an extension of the heuristic pseudoknot prediction algorithm DotKnot, which covers H-type pseudoknots and intramolecular kissing hairpins. Our framework allows for easy integration of advanced H-type pseudoknot energy models. For a test set of RNA sequences containing kissing hairpins and other types of pseudoknot structures, DotKnot outperforms competing methods from the literature. DotKnot is available as a web server under http://dotknot.csse.uwa.edu.au.

Efficient preparation of internally modified single-molecule constructs using nicking enzymes

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required.

The Osmolyte TMAO Stabilizes Native RNA Tertiary Structures in the Absence of Mg2+: Evidence for a Large Barrier to Folding from Phosphate Dehydration

The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ∼0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol−1m−1 for four RNA tertiary structures (30–86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg2+ and that the structures stabilized by TMAO and Mg2+ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg2+ ion chelation site formed in part by a buried phosphate, even though Mg2+ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, +211 cal mol−1m−1 for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66–173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg2+ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.

Alternative Ways to Think about Cellular Internal Ribosome Entry

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of "cap-dependent" and "IRES-dependent" should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation.

Predicting Ion Binding Properties for RNA Tertiary Structures

Recent experiments pointed to the potential importance of ion correlation for multivalent ions such as Mg2+ ions in RNA folding. In this study, we develop an all-atom model to predict the ion electrostatics in RNA folding. The model can treat ion correlation effects explicitly by considering an ensemble of discrete ion distributions. In contrast to the previous coarse-grained models that can treat ion correlation, this new model is based on all-atom nucleic acid structures. Thus, unlike the previous coarse-grained models, this new model allows us to treat complex tertiary structures such as HIV-1 DIS type RNA kissing complexes. Theory-experiment comparisons for a variety of tertiary structures indicate that the model gives improved predictions over the Poisson-Boltzmann theory, which underestimates the Mg2+ binding in the competition with Na+. Further systematic theory-experiment comparisons for a series of tertiary structures lead to a set of analytical formulas for Mg2+/Na+ ion-binding to various RNA and DNA structures over a wide range of Mg2+ and Na+ concentrations.

Multistage collapse of a bacterial ribozyme observed by time-resolved small-angle X-ray scattering.

Ribozymes must fold into compact, native structures to function properly in the cell. The first step in forming the RNA tertiary structure is the neutralization of the phosphate charge by cations, followed by collapse of the unfolded molecules into more compact structures. The specificity of the collapse transition determines the structures of the folding intermediates and the folding time to the native state. However, the forces that enable specific collapse in RNA are not understood. Using time-resolved SAXS, we report that upon addition of 5 mM Mg(2+) to the Azoarcus group I ribozyme up to 80% of chains form compact structures in less than 1 ms. In 1 mM Mg(2+), the collapse transition produces extended structures that slowly approach the folded state, while > or = 1.5 mM Mg(2+) leads to an ensemble of random coils that fold with multistage kinetics. Increased flexibility of molecules in the intermediate ensemble correlates with a Mg(2+)-dependent increase in the fast folding population and a previously unobserved crossover in the collapse kinetics. Partial denaturation of the unfolded RNA with urea also increases the fraction of chains following the fast-folding pathway. These results demonstrate that the preferred collapse mechanism depends on the extent of Mg(2+)-dependent charge neutralization and that non-native interactions within the unfolded ensemble contribute to the heterogeneity of the ribozyme folding pathways at the very earliest stages of tertiary structure formation.

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

To what extent small differences in RNA sequences (mutations) can have a profound impact on biology remains an intriguing question. This effect can be studied by using untranslated RNA regions as a model. We have studied the influence of mutations on the structure of an RNA hairpin that occurs in the 3'-untranslated region (UTR) of Flaviviridae, and is known to have a large impact on the vector dependency of flaviviruses. Three related RNA sequences were studied by NMR spectroscopy. The selected sequences represent each one of the three clusters in the flavivirus genes (mosquito-borne, tick-borne, and no-known-vector viruses). A new strategy was used to obtain chemical shift signatures of carbonyl atoms in unlabeled uridine nucleobases to characterize their involvement in hydrogen bonding. Clear differences occur in the structures and stacking pattern of the three RNA hairpins. The observed differences cannot be predicted based on sequence analysis. A different biology can be correlated with a different RNA tertiary structure. The underlying biological mechanism, however, remains to be studied.

Precise Mapping of RNA Tertiary Structure via Nanometer Distance Measurements with Double Electron−Electron Resonance Spectroscopy

Divalent metal (Mg(2+)) ion-dependent folding of the hammerhead ribozyme from Schistosoma mansoni was monitored with double electron-electron resonance (DEER) pulsed electron paramagnetic resonance spectroscopy by measuring nanometer-scale distances between paramagnetic spin-labels attached to the RNA. DEER measurements detect global folding of the ribozyme with excellent agreement between predictions from experimental, modeled, and crystallographic measurements. These measurements demonstrate the use of DEER spectroscopy as a tool for structural analysis of complex RNAs.

Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs

The heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in the regulation of mRNA metabolism by specifically recognizing G-tract RNA sequences. We have determined the solution structures of the three quasi-RNA-recognition motifs (qRRMs) of hnRNP F in complex with G-tract RNA. These structures show that qRRMs bind RNA in a very unusual manner, with the G-tract 'encaged', making the qRRM a novel RNA binding domain. We defined a consensus signature sequence for qRRMs and identified other human qRRM-containing proteins that also specifically recognize G-tract RNAs. Our structures explain how qRRMs can sequester G-tracts, maintaining them in a single-stranded conformation. We also show that isolated qRRMs of hnRNP F are sufficient to regulate the alternative splicing of the Bcl-x pre-mRNA, suggesting that hnRNP F would act by remodeling RNA secondary and tertiary structures.

A Probabilistic and Continuous Model of Protein Conformational Space for Template-Free Modeling

One of the major challenges with protein template-free modeling is an efficient sampling algorithm that can explore a huge conformation space quickly. The popular fragment assembly method constructs a conformation by stringing together short fragments extracted from the Protein Data Base (PDB). The discrete nature of this method may limit generated conformations to a subspace in which the native fold does not belong. Another worry is that a protein with really new fold may contain some fragments not in the PDB. This article presents a probabilistic model of protein conformational space to overcome the above two limitations. This probabilistic model employs directional statistics to model the distribution of backbone angles and 2(nd)-order Conditional Random Fields (CRFs) to describe sequence-angle relationship. Using this probabilistic model, we can sample protein conformations in a continuous space, as opposed to the widely used fragment assembly and lattice model methods that work in a discrete space. We show that when coupled with a simple energy function, this probabilistic method compares favorably with the fragment assembly method in the blind CASP8 evaluation, especially on alpha or small beta proteins. To our knowledge, this is the first probabilistic method that can search conformations in a continuous space and achieves favorable performance. Our method also generated three-dimensional (3D) models better than template-based methods for a couple of CASP8 hard targets. The method described in this article can also be applied to protein loop modeling, model refinement, and even RNA tertiary structure prediction.