Alternative RNA structures formed during transcription depend on elongation rate and modify RNA processing.

Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing and determined how the "nascent RNA structureome" relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3' splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV, which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence, co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary, the plasticity of nascent transcripts has widespread effects on RNA processing.

The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription.

The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.

Structure-Based Mechanisms of a Molecular RNA Polymerase/Chaperone Machine Required for Ribosome Biosynthesis.

Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.

Structural basis of rotavirus RNA chaperone displacement and RNA annealing.

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

Cis regulation within a cluster of viral microRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.

The Sac10b homolog from Sulfolobus islandicus is an RNA chaperone.

Nucleic acid-binding proteins of the Sac10b family, also known as Alba, are widely distributed in Archaea. However, the physiological roles of these proteins have yet to be clarified. Here, we show that Sis10b, a member of the Sac10b family from the hyperthermophilic archaeon Sulfolobus islandicus, was active in RNA strand exchange, duplex RNA unwinding in vitro and RNA unfolding in a heterologous host cell. This protein exhibited temperature-dependent binding preference for ssRNA over dsRNA and was more efficient in RNA unwinding and RNA unfolding at elevated temperatures. Notably, alanine substitution of a highly conserved basic residue (K) at position 17 in Sis10b drastically reduced the ability of this protein to catalyse RNA strand exchange and RNA unwinding. Additionally, the preferential binding of Sis10b to ssRNA also depended on the presence of K17 or R17. Furthermore, normal growth was restored to a slow-growing Sis10b knockdown mutant by overproducing wild-type Sis10b but not by overproducing K17A in this mutant strain. Our results indicate that Sis10b is an RNA chaperone that likely functions most efficiently at temperatures optimal for the growth of S. islandicus, and K17 is essential for the chaperone activity of the protein.

Same fold, different properties: polarizable molecular dynamics simulations of telomeric and TERRA G-quadruplexes.

DNA and RNA sequences rich in guanine can fold into noncanonical structures called G-quadruplexes (GQs), which exhibit a common stem structure of Hoogsteen hydrogen-bonded guanine tetrads and diverse loop structures. GQ sequence motifs are overrepresented in promoters, origins of replication, telomeres, and untranslated regions in mRNA, suggesting roles in modulating gene expression and preserving genomic integrity. Given these roles and unique aspects of different structures, GQs are attractive targets for drug design, but greater insight into GQ folding pathways and the interactions stabilizing them is required. Here, we performed molecular dynamics simulations to study two bimolecular GQs, a telomeric DNA GQ and the analogous telomeric repeat-containing RNA (TERRA) GQ. We applied the Drude polarizable force field, which we show outperforms the additive CHARMM36 force field in both ion retention and maintenance of the GQ folds. The polarizable simulations reveal that the GQs bind bulk K+ ions differently, and that the TERRA GQ accumulates more K+ ions, suggesting different ion interactions stabilize these structures. Nucleobase dipole moments vary as a function of position and also contribute to ion binding. Finally, we show that the TERRA GQ is more sensitive than the telomeric DNA GQ to water-mediated modulation of ion-induced dipole-dipole interactions.

METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis.

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

Efficient inhibition of RNA self-primed extension by addition of competing 3'-capture DNA-improved RNA synthesis by T7 RNA polymerase.

In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as RNAs with lengths shorter and longer than the expected runoff RNA. We have recently confirmed that longer RNA products are formed predominantly via cis self-primed extension, in which released runoff RNA folds back on itself to prime its own RNA-templated extension. In the current work, we demonstrate that addition of a DNA oligonucleotide (capture DNA) that is complementary to the 3' end of the expected runoff RNA effectively prevents self-primed extension, even under conditions commonly used for high RNA yields. Moreover, the presence of this competing capture DNA during 'high yield' transcription, leads to an increase in the yield of expected runoff RNA by suppressing the formation of undesired longer RNA byproducts.

A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure.

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

Complex Conformational Dynamics of the Heart Failure-Associated Pre-miRNA-377 Hairpin Revealed by Single-Molecule Optical Tweezers.

Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.

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.

Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo.

RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo.

A functional genetic screen reveals sequence preferences within a key tertiary interaction in cobalamin riboswitches required for ligand selectivity.

Riboswitches are structured mRNA sequences that regulate gene expression by directly binding intracellular metabolites. Generating the appropriate regulatory response requires the RNA rapidly and stably acquire higher-order structure to form the binding pocket, bind the appropriate effector molecule and undergo a structural transition to inform the expression machinery. These requirements place riboswitches under strong kinetic constraints, likely restricting the sequence space accessible by recurrent structural modules such as the kink turn and the T-loop. Class-II cobalamin riboswitches contain two T-loop modules: one directing global folding of the RNA and another buttressing the ligand binding pocket. While the T-loop module directing folding is highly conserved, the T-loop associated with binding is substantially less so, with no clear consensus sequence. To further understand the functional role of the binding-associated module, a functional genetic screen of a library of riboswitches with the T-loop and its interacting nucleotides was used to build an experimental phylogeny comprised of sequences that possess a wide range of cobalamin-dependent regulatory activity. Our results reveal conservation patterns of the T-loop and its interaction with the binding core that allow for rapid tertiary structure formation and demonstrate its importance for generating strong ligand-dependent repression of mRNA expression.

Tiling Nussinov's RNA folding loop nest with a space-time approach.

BACKGROUND: An RNA primary structure, or sequence, is a single strand considered as a chain of nucleotides from the alphabet AUGC (adenine, uracil, guanine, cytosine). The strand can be folded onto itself, i.e., one segment of an RNA sequence might be paired with another segment of the same RNA sequence into a two-dimensional structure composed by a list of complementary base pairs, which are close together with the minimum energy. That list is called RNA's secondary structure and is predicted by an RNA folding algorithm. RNA secondary structure prediction is a computing-intensive task that lies at the core of search applications in bioinformatics. RESULTS: We suggest a space-time tiling approach and apply it to generate parallel cache effective tiled code for RNA folding using Nussinov's algorithm. CONCLUSIONS: Parallel tiled code generated with a suggested space-time loop tiling approach outperforms known related codes generated automatically by means of optimizing compilers and codes produced manually. The presented approach enables us to tile all the three loops of Nussinov's recurrence that is not possible with commonly known tiling techniques. Generated parallel tiled code is scalable regarding to the number of parallel threads - increasing the number of threads reduces code execution time. Defining speed up as the ratio of the time taken to run the original serial program on one thread to the time taken to run the tiled program on P threads, we achieve super-linear speed up (a value of speed up is greater than the number of threads used) for parallel tiled code against the original serial code up to 32 threads and super-linear speed up scalability (increasing speed up with increasing the thread number) up to 8 threads. For one thread used, speed up is about 4.2 achieved on an Intel Xeon machine used for carrying out experiments.

Enhancing heterologous expression in Chlamydomonas reinhardtii by transcript sequence optimization.

Various species of microalgae have recently emerged as promising host-organisms for use in biotechnology industries due to their unique properties. These include efficient conversion of sunlight into organic compounds, the ability to grow in extreme conditions and the occurrence of numerous post-translational modification pathways. However, the inability to obtain high levels of nuclear heterologous gene expression in microalgae hinders the development of the entire field. To overcome this limitation, we analyzed different sequence optimization algorithms while studying the effect of transcript sequence features on heterologous expression in the model microalga Chlamydomonas reinhardtii, whose genome consists of rare features such as a high GC content. Based on the analysis of genomic data, we created eight unique sequences coding for a synthetic ferredoxin-hydrogenase enzyme, used here as a reporter gene. Following in silico design, these synthetic genes were transformed into the C. reinhardtii nucleus, after which gene expression levels were measured. The empirical data, measured in vivo show a discrepancy of up to 65-fold between the different constructs. In this work we demonstrate how the combination of computational methods and our empirical results enable us to learn about the way gene expression is encoded in the C. reinhardtii transcripts. We describe the deleterious effect on overall expression of codons encoding for splicing signals. Subsequently, our analysis shows that utilization of a frequent subset of preferred codons results in elevated transcript levels, and that mRNA folding energy in the vicinity of translation initiation significantly affects gene expression.

Identification of the RNA Pseudoknot within the 3' End of the Porcine Reproductive and Respiratory Syndrome Virus Genome as a Pathogen-Associated Molecular Pattern To Activate Antiviral Signaling via RIG-I and Toll-Like Receptor 3.

Once infected by viruses, cells can detect pathogen-associated molecular patterns (PAMPs) on viral nucleic acid by host pattern recognition receptors (PRRs) to initiate the antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory diseases in pigs of different ages. To date, the sensing mechanism of PRRSV has not been elucidated. Here, we reported that the pseudoknot region residing in the 3' untranslated regions (UTR) of the PRRSV genome, which has been proposed to regulate RNA synthesis and virus replication, was sensed as nonself by retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3) and strongly induced type I interferons (IFNs) and interferon-stimulated genes (ISGs) in porcine alveolar macrophages (PAMs). The interaction between the two stem-loops inside the pseudoknot structure was sufficient for IFN induction, since disruption of the pseudoknot interaction powerfully dampened the IFN induction. Furthermore, transfection of the 3' UTR pseudoknot transcripts in PAMs inhibited PRRSV replication in vitro Importantly, the predicted similar structures of other arterivirus members, including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV), also displayed strong IFN induction activities. Together, in this work we identified an innate recognition mechanism by which the PRRSV 3' UTR pseudoknot region served as PAMPs of arteriviruses and activated innate immune signaling to produce IFNs that inhibit virus replication. All of these results provide novel insights into innate immune recognition during virus infection.IMPORTANCE PRRS is the most common viral disease in the pork industry. It is caused by PRRSV, a positive single-stranded RNA virus, whose infection often leads to persistent infection. To date, it is not yet clear how PRRSV is recognized by the host and what is the exact mechanism of IFN induction. Here, we investigated the nature of PAMPs on PRRSV and the associated PRRs. We found that the 3' UTR pseudoknot region of PRRSV, which has been proposed to regulate viral RNA synthesis, could act as PAMPs recognized by RIG-I and TLR3 to induce type I IFN production to suppress PRRSV infection. This report is the first detailed description of pattern recognition for PRRSV, which is important in understanding the antiviral response of arteriviruses, especially PRRSV, and extends our knowledge on virus recognition.

Kinetics coming into focus: single-molecule microscopy of riboswitch dynamics.

Riboswitches are dynamic RNA motifs that are mostly embedded in the 5'-untranslated regions of bacterial mRNAs, where they regulate gene expression transcriptionally or translationally by undergoing conformational changes upon binding of a small metabolite or ion. Due to the small size of typical ligands, relatively little free energy is available from ligand binding to overcome the often high energetic barrier of reshaping RNA structure. Instead, most riboswitches appear to take advantage of the directional and hierarchical folding of RNA by employing the ligand as a structural 'linchpin' to adjust the kinetic partitioning between alternate folds. In this model, even small, local structural and kinetic effects of ligand binding can cascade into global RNA conformational changes affecting gene expression. Single-molecule (SM) microscopy tools are uniquely suited to study such kinetically controlled RNA folding since they avoid the ensemble averaging of bulk techniques that loses sight of unsynchronized, transient, and/or multi-state kinetic behavior. This review summarizes how SM methods have begun to unravel riboswitch-mediated gene regulation.

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5'-untraslated region.

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

TurboFold II: RNA structural alignment and secondary structure prediction informed by multiple homologs.

This paper presents TurboFold II, an extension of the TurboFold algorithm for predicting secondary structures for multiple RNA homologs. TurboFold II augments the structure prediction capabilities of TurboFold by additionally providing multiple sequence alignments. Probabilities for alignment of nucleotide positions between all pairs of input sequences are iteratively estimated in TurboFold II by incorporating information from both the sequence identity and secondary structures. A multiple sequence alignment is obtained from these probabilities by using a probabilistic consistency transformation and a hierarchically computed guide tree. To assess TurboFold II, its sequence alignment and structure predictions were compared with leading tools, including methods that focus on alignment alone and methods that provide both alignment and structure prediction. TurboFold II has comparable alignment accuracy with MAFFT and higher accuracy than other tools. TurboFold II also has comparable structure prediction accuracy as the original TurboFold algorithm, which is one of the most accurate methods. TurboFold II is part of the RNAstructure software package, which is freely available for download at http://rna.urmc.rochester.edu under a GPL license.