Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5'GGUC3' paired to 3'CUGG5' separately from other 5'GU3'/3'UG5' stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

Challenges and approaches to predicting RNA with multiple functional structures.

The revolution in sequencing technology demands new tools to interpret the genetic code. As in vivo transcriptome-wide chemical probing techniques advance, new challenges emerge in the RNA folding problem. The emphasis on one sequence folding into a single minimum free energy structure is fading as a new focus develops on generating RNA structural ensembles and identifying functional structural features in ensembles. This review describes an efficient combinatorially complete method and three free energy minimization approaches to predicting RNA structures with more than one functional fold, as well as two methods for analysis of a thermodynamics-based Boltzmann ensemble of structures. The review then highlights two examples of viral RNA 3'-UTR regions that fold into more than one conformation and have been characterized by single molecule fluorescence energy resonance transfer or NMR spectroscopy. These examples highlight the different approaches and challenges in predicting structure and function from sequence for RNA with multiple biological roles and folds. More well-defined examples and new metrics for measuring differences in RNA structures will guide future improvements in prediction of RNA structure and function from sequence.

Thermodynamic stabilities of three-way junction nanomotifs in prohead RNA.

The thermodynamic stabilities of four natural prohead or packaging RNA (pRNA) three-way junction (3WJ) nanomotifs and seven phi29 pRNA 3WJ deletion mutant nanomotifs were investigated using UV optical melting on a three-component RNA system. Our data reveal that some pRNA 3WJs are more stable than the phi29 pRNA 3WJ. The stability of the 3WJ contributes to the unique self-assembly properties of pRNA. Thus, ultrastable pRNA 3WJ motifs suggest new scaffolds for pRNA-based nanotechnology. We present data demonstrating that pRNA 3WJs differentially respond to the presence of metal ions. A comparison of our data with free energies predicted by currently available RNA secondary structure prediction programs shows that these programs do not accurately predict multibranch loop stabilities. These results will expand the existing parameters used for RNA secondary structure prediction from sequence in order to better inform RNA structure-function hypotheses and guide the rational design of functional RNA supramolecular assemblies.

Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops.

Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites.

Surprising Sequence Effects on GU Closure of Symmetric 2 × 2 Nucleotide RNA Internal Loops.

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

Swellix: a computational tool to explore RNA conformational space.

BACKGROUND: The sequence of nucleotides in an RNA determines the possible base pairs for an RNA fold and thus also determines the overall shape and function of an RNA. The Swellix program presented here combines a helix abstraction with a combinatorial approach to the RNA folding problem in order to compute all possible non-pseudoknotted RNA structures for RNA sequences. The Swellix program builds on the Crumple program and can include experimental constraints on global RNA structures such as the minimum number and lengths of helices from crystallography, cryoelectron microscopy, or in vivo crosslinking and chemical probing methods. RESULTS: The conceptual advance in Swellix is to count helices and generate all possible combinations of helices rather than counting and combining base pairs. Swellix bundles similar helices and includes improvements in memory use and efficient parallelization. Biological applications of Swellix are demonstrated by computing the reduction in conformational space and entropy due to naturally modified nucleotides in tRNA sequences and by motif searches in Human Endogenous Retroviral (HERV) RNA sequences. The Swellix motif search reveals occurrences of protein and drug binding motifs in the HERV RNA ensemble that do not occur in minimum free energy or centroid predicted structures. CONCLUSIONS: Swellix presents significant improvements over Crumple in terms of efficiency and memory use. The efficient parallelization of Swellix enables the computation of sequences as long as 418 nucleotides with sufficient experimental constraints. Thus, Swellix provides a practical alternative to free energy minimization tools when multiple structures, kinetically determined structures, or complex RNA-RNA and RNA-protein interactions are present in an RNA folding problem.

Probing viral genomic structure: alternative viewpoints and alternative structures for satellite tobacco mosaic virus RNA.

Viral RNA structure prediction is a valuable tool for development of drugs against viral disease. This work discusses different approaches to predicting encapsidated viral RNA and highlights satellite tobacco mosaic virus (STMV) RNA as a model system with excellent crystallography data. Fundamentally important issues for debate include thermodynamic versus kinetic control of virus assembly and the possible consequences of quasi-species in the primary structure on RNA secondary structure prediction of a single structure or an ensemble of structures. Multiple computational tools and chemical reagents are now available for improved viral RNA structure prediction. Two different predicted structures for encapsidated STMV RNA result from differences in three main areas: a different approach and philosophy to studying encapsidated viral RNA, an emphasis on different RNA motifs, and technical differences in computational methods and chemical reagents. The experiments with traditional chemical probing and SHAPE reagents are compared in terms of chemistry, results, and interpretation for STMV RNA as well as other RNA protein assemblies, such as the 5'UTR of HIV and the ribosome. This discussion of the challenges of viral RNA structure prediction will lead to new experiments and improved future predictions for viral RNA.

Incorporating global features of RNA motifs in predictions for an ensemble of secondary structures for encapsidated MS2 bacteriophage RNA.

The secondary structure of encapsidated MS2 genomic RNA poses an interesting RNA folding challenge. Cryoelectron microscopy has demonstrated that encapsidated MS2 RNA is well-ordered. Models of MS2 assembly suggest that the RNA hairpin-protein interactions and the appropriate placement of hairpins in the MS2 RNA secondary structure can guide the formation of the correct icosahedral particle. The RNA hairpin motif that is recognized by the MS2 capsid protein dimers, however, is energetically unfavorable, and thus free energy predictions are biased against this motif. Computer programs called Crumple, Sliding Windows, and Assembly provide useful tools for prediction of viral RNA secondary structures when the traditional assumptions of RNA structure prediction by free energy minimization may not apply. These methods allow incorporation of global features of the RNA fold and motifs that are difficult to include directly in minimum free energy predictions. For example, with MS2 RNA the experimental data from SELEX experiments, crystallography, and theoretical calculations of the path for the series of hairpins can be incorporated in the RNA structure prediction, and thus the influence of free energy considerations can be modulated. This approach thoroughly explores conformational space and generates an ensemble of secondary structures. The predictions from this new approach can test hypotheses and models of viral assembly and guide construction of complete three-dimensional models of virus particles.

Nanopore Direct RNA Sequencing Reveals Virus-Induced Changes in the Transcriptional Landscape in Human Bronchial Epithelial Cells.

Direct RNA nanopore sequencing reveals changes in gene expression, polyadenylation, splicing, m6A methylation, and pseudouridylation in response to influenza virus exposure in primary human bronchial epithelial cells. This study focuses on the epitranscriptomic profile of genes in the host immune response. In addition to polyadenylated noncoding RNA, we purified and sequenced nonpolyadenylated noncoding RNA and observed changes in expression, N6-methyl-adenosine (m6A), and pseudouridylation (Ψ) in these novel RNA. Two recently discovered lincRNA with roles in immune response, Chaserr and LEADR , became highly methylated in response to influenza exposure. Several H/ACA type snoRNAs that guide pseudouridylation are decreased in expression in response to influenza, and there is a corresponding decrease in the pseudouridylation of two novel lncRNA. Thus, novel epitranscriptomic changes revealed by direct RNA sequencing with nanopore technology provides unique insights into the host epitranscriptomic changes in epithelial gene networks that respond to influenza virus infection.

Different sequences show similar quaternary interaction stabilities in prohead viral RNA self-assembly.

Prohead RNA (pRNA) is an essential component of the self-assembling ϕ29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.

Ensemble of secondary structures for encapsidated satellite tobacco mosaic virus RNA consistent with chemical probing and crystallography constraints.

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated. The high-resolution crystallographic structure of the satellite tobacco mosaic virus (STMV) particle reveals 30 helices of well-ordered RNA. The crystallographic data provide global constraints on the possible secondary structures for the encapsidated RNA. Traditional free energy minimization methods of RNA secondary structure prediction do not generate structures consistent with the crystallographic data, and to date no complete STMV RNA basepaired secondary structure has been generated. RNA-protein interactions and tertiary interactions may contribute a significant degree of stability, and the kinetics of viral assembly may dominate the folding process. The computational tools, Helix Find & Combine, Crumple, and Sliding Windows and Assembly, evaluate and explore the possible secondary structures for encapsidated STMV RNA. All possible hairpins consistent with the experimental data and a cotranscriptional folding and assembly hypothesis were generated, and the combination of hairpins that was most consistent with experimental data is presented as the best representative structure of the ensemble. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold.

Nuclear magnetic resonance structure of the prohead RNA E-loop hairpin.

The Bacillus subtilis phage phi29 packaging motor requires prohead RNA for genome encapsidation. The nuclear magnetic resonance structure of the prohead RNA E-loop hairpin, r(5'AUUGAGUU), is presented and compared to predictions from MC-SYM. The prohead RNA E-loop hairpins contain sequences similar to rRNA hairpins. Comparison of predicted and experimentally determined prohead and ribosomal hairpin structures reveals that sequence similarity is a stronger determinant of hairpin structural similarity than grouping similar types of RNA. All the hairpins contain a U-turn motif but differ in the first noncanonical pair and backbone orientation. These structures provide benchmarks for further improvements in RNA structure predictions from sequence.

Consecutive terminal GU pairs stabilize RNA helices.

Consecutive GU pairs at the ends of RNA helices provide significant thermodynamic stability between -1.0 and -3.8 kcal/mol at 37 °C, which is equivalent to approximately 2 orders of magnitude in the value of a binding constant. The thermodynamic stabilities of GU pairs depend on the sequence, stacking orientation, and position in the helix. In contrast to GU pairs in the middle of a helix that may be destabilizing, all consecutive terminal GU pairs contribute favorable thermodynamic stability. This work presents measured thermodynamic stabilities for 30 duplexes containing two, three, or four consecutive GU pairs at the ends of RNA helices and a model to predict the thermodynamic stabilities of terminal GU pairs. Imino proton NMR spectra show that the terminal GU nucleotides form hydrogen-bonded pairs. Different orientations of terminal GU pairs can have different conformations with equivalent thermodynamic stabilities. These new data and prediction model will help improve RNA secondary structure prediction, identification of miRNA target sequences with GU pairs, and efforts to understand the fundamental physical forces directing RNA structure and energetics.

Perspectives on Viral RNA Genomes and the RNA Folding Problem.

Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.

3' terminal nucleotides determine thermodynamic stabilities of mismatches at the ends of RNA helices.

The thermodynamic stabilities of consecutive mismatches at the ends of RNA helices are determined by the 3' terminal nucleotides. More than 40 RNA duplexes containing terminal motifs of 3 or more nucleotides were studied by optical melting experiments. Up to three noncanonical pairs of nucleotides at the end of RNA helices provide additional thermodynamic stability. 3' nucleotides contribute more stability than 5' nucleotides, and purines contribute more stability than pyrimidines. The additional stability of a second or third 3' nucleotide stacking on a purine is the same for both dangling ends and consecutive terminal mismatches. Current predictions underestimate RNA duplex stabilities with terminal motifs by 1.4 kcal/mol on average, which is an order of magnitude in a binding constant at 37 degrees C. Accurate thermodynamic parameters for these terminal motifs will contribute to improvements in RNA secondary structure predictions, identification of microRNA targets, and design of siRNA therapeutics with fewer off-target effects.

The structures of antibiotics bound to the E site region of the 50 S ribosomal subunit of Haloarcula marismortui: 13-deoxytedanolide and girodazole.

Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.

Crumple: An Efficient Tool to Explore Thoroughly the RNA Folding Landscape.

The folding landscape for an RNA sequence contains many diverse structures and motifs, which are often sampled rather than completely explored. Today's supercomputers make the complete enumeration of all possible folds for an RNA and a detailed description of the RNA folding landscape a more feasible task. This chapter provides protocols for using the Crumple folding algorithm, an efficient tool to generate all possible non-pseudoknotted folds for an RNA sequence. Crumple in conjunction with Sliding Windows and Assembly can incorporate experimental constraints on the global features of an RNA, such as the minimum number and lengths of helices, which may be determined by crystallography or cryo-electron microscopy. This complete enumeration method is independent of free-energy minimization and allows the user to incorporate experimental data such as chemical probing, SELEX data on RNA-protein binding motifs, and phylogenetic covariation.