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Improving RNA Secondary Structure Prediction Through Expanded Training Data.

In recent years, deep learning has revolutionized protein structure prediction, achieving remarkable speed and accuracy. RNA structure prediction, however, has lagged behind. Although several methods have shown some success in predicting RNA secondary and tertiary structures, none have reached the accuracy observed with contemporary protein models. The lack of success of these RNA structure prediction models has been proposed to be due to limited high-quality structural information that can be used as training data. To probe this proposed limitation, we developed a large and diverse dataset comprising paired RNA sequences and their corresponding secondary structures. We assess the utility of this enhanced dataset by retraining on a deep learning model, SincFold. We find that SincFold exhibited improved generalization to some previously unseen RNA families, enhancing its capability to predict accurate de novo RNA secondary structures. The RNASSTR dataset provides a substantial advance for RNA structure modeling, laying a strong foundation for the development of future RNA secondary structure prediction algorithms.

Journal Article↗

Statistics of RNA secondary structures.

A statistical reference for RNA secondary structures with minimum free energies is computed by folding large ensembles of random RNA sequences. Four nucleotide alphabets are used: two binary alphabets, AU and GC, the biophysical AUGC and the synthetic GCXK alphabet. RNA secondary structures are made of structural elements, such as stacks, loops, joints, and free ends. Statistical properties of these elements are computed for small RNA molecules of chain lengths up to 100. The results of RNA structure statistics depend strongly on the particular alphabet chosen. The statistical reference is compared with the data derived from natural RNA molecules with similar base frequencies. Secondary structures are represented as trees. Tree editing provides a quantitative measure for the distance dt, between two structures. We compute a structure density surface as the conditional probability of two structures having distance t given that their sequences have distance h. This surface indicates that the vast majority of possible minimum free energy secondary structures occur within a fairly small neighborhood of any typical (random) sequence. Correlation lengths for secondary structures in their tree representations are computed from probability densities. They are appropriate measures for the complexity of the sequence-structure relation. The correlation length also provides a quantitative estimate for the mean sensitivity of structures to point mutations.

Base Sequence↗

Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.

Base Composition↗

High salt solution structure of a left-handed RNA double helix.

Right-handed RNA duplexes of (CG)n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)2 determined in 6 M NaClO4. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6(n)-H5'/H5''(n)-H8(n+1)-H1'(n+1)-H6(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4-7 degrees), whereas GpC steps have large negative values (-61 degrees). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2'-OH groups, and the major groove are well defined. The 2'-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2'-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.

Base Pairing↗

A computational procedure for assessing the significance of RNA secondary structure.

In our recent series of papers, we have used the structures of statistical significance from Monte Carlo simulations to improve the predictions of secondary structure of RNA and to analyze the possible role of locally significant structures in the life cycle of human immunodeficiency virus. Because of intensive computational requirements for Monte Carlo simulation, it becomes impractical even using a supercomputer to assess the significance of a structure with a window size greater than 200 along an RNA sequence of 1000 bases or more. In this paper, we have developed a new procedure that drastically reduces the time needed to assess the significance of structures. In fact, the efficiency of this new method allows us to assess structures on the VAX as well as the CRAY.

Biometry↗

A kinetic approach to the prediction of RNA secondary structures.

A new approach to the prediction of secondary RNA structures based on the analysis of the kinetics of molecular self-organisation is proposed herein. The Markov process is used to describe structural reconstructions during secondary structure formation. This process is modelled by a Monte-Carlo method. Examples of the calculation by this method of the secondary structures kinetic ensemble are given. Distribution of time-dependent probabilities within the ensembles is obtained. An effective method for search for the equilibrium ensemble is also suggested. This method is based on the construction of a tree of all possible secondary structures of RNA. By ascribing a probability for each structure (according to its free energy) the Boltzmann equilibrium ensemble can be obtained.

Computers↗

The effect of RNA secondary structure on the action of a nucleolar endoribonuclease.

The effect of the folded macromolecular structure of RNA on the action of a purified single strand specific nucleolar ribonuclease was studied by comparing the limited hydrolysis of defined RNA substrates. The nucleolar RNase was shown to attack only single-stranded regions of the native 5.8 S rRNA, consistent with a computer-derived model for the secondary structure (Nazar, R. N., Stitz, T. O., and Busch, H. (1975) J. Biol. Chem. 250, 8591-8597). The single strand specific nucleolar RNase, unlike S1 nuclease, does not release the end-labeled nucleotide and therefore provides a more useful probe for structural analysis at or near 3'- or 5'-terminal ends of an RNA molecule. Although attacking only single strand regions of the native 5.8 S rRNA, the selectivity of the nucleolar RNase, when compared to S1 nuclease, is distinct and supports the suggestion that other factors besides the proposed secondary structure must also influence nuclease attack. The selective and limited attack of the nucleolar RNase on native RNA was similarly observed using mouse 45 S preribosomal RNA as a substrate and possibly suggests a role for this nucleolar RNase in ribosomal RNA processing.

Animals↗

Hybridization of two oligodeoxynucleotides to both strands of an RNA hairpin structure increases the efficiency of RNA-DNA duplex formation.

Hybridization of two oligodeoxyribonucleotides (ON1 and ON2), complementary to opposite strands of the apical domain of Escherichia coli 4.5S RNA, was studied. ON1, complementary to bases 58-71, was not able to form a stable RNA-DNA hybrid whereas ON2, complementary to bases 38-53, was. Addition of both oligonucleotides at the same time resulted in the formation of a ternary complex permitting hybridization of ON1 and increasing hybridization of ON2. Under this condition, binary complexes of ON1 or ON2 with 4.5S RNA were not observed. The data demonstrate that hybridization of oligonucleotides to both strands of an RNA hairpin structure increases the efficiency of hybridization of either oligonucleotide.

Base Sequence↗

RNA ligase structures reveal the basis for RNA specificity and conformational changes that drive ligation forward.

T4 RNA ligase 2 (Rnl2) and kinetoplastid RNA editing ligases exemplify a family of RNA repair enzymes that seal 3'OH/5'PO(4) nicks in duplex RNAs via ligase adenylylation (step 1), AMP transfer to the nick 5'PO(4) (step 2), and attack by the nick 3'OH on the 5'-adenylylated strand to form a phosphodiester (step 3). Crystal structures are reported for Rnl2 at discrete steps along this pathway: the covalent Rnl2-AMP intermediate; Rnl2 bound to an adenylylated nicked duplex, captured immediately following step 2; and Rnl2 at an adenylylated nick in a state poised for step 3. These structures illuminate the stereochemistry of nucleotidyl transfer and reveal how remodeling of active-site contacts and conformational changes propel the ligation reaction forward. Mutational analysis and comparison of nick-bound structures of Rnl2 and human DNA ligase I highlight common and divergent themes of substrate recognition that can explain their specialization for RNA versus DNA repair.

Adenosine Monophosphate↗

Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production.

RNA editing of the hepatitis delta virus (HDV) antigenome at the amber/W site by the host RNA adenosine deaminase ADAR1 is a critical step in the HDV replication cycle. Editing is required for production of the viral protein hepatitis delta antigen long form (HDAg-L), which is necessary for viral particle production but can inhibit HDV RNA replication. The RNA secondary structural features in ADAR1 substrates are not completely defined, but base pairing in the 20-nucleotide (nt) region 3' of editing sites is thought to be important. The 25-nt region 3' of the HDV amber/W site in HDV genotype I RNA consists of a conserved secondary structure that is mostly base paired but also has asymmetric internal loops and single-base bulges. To understand the effect of this 3' region on the HDV replication cycle, mutations that either increase or decrease base pairing in this region were created and the effects of these changes on amber/W site editing, RNA replication, and virus production were studied. Increased base pairing, particularly in the region 15 to 25 nt 3' of the editing site, significantly increased editing; disruption of base pairing in this region had little effect. Increased editing resulted in a dramatic inhibition of HDV RNA synthesis, mostly due to excess HDAg-L production. Although virus production at early times was unaffected by this reduced RNA replication, at later times it was significantly reduced. Therefore, it appears that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efficiency but for optimal viral replication and secretion.

Adenosine Deaminase↗

Measuring the thermodynamics of RNA secondary structure formation.

The thermodynamics of RNA secondary structure formation in small model systems provides a database for predicting RNA structure from sequence. Methods for making these measurements are reviewed with emphasis on optical methods and treatment of experimental errors. Analysis of experimental results in terms of simple nearest-neighbor models is presented. Some measured sequence dependences of non-Watson-Crick motifs are discussed.

Base Composition↗

Comparative sequence analysis and patterns of covariation in RNA secondary structures.

A novel method of RNA secondary structure prediction based on a comparison of nucleotide sequences is described. This method correctly predicts nearly all evolutionarily conserved secondary structures of five different RNAs: tRNA, 5S rRNA, bacterial ribonuclease P (RNase P) RNA, eukaryotic small subunit rRNA, and the 3' untranslated region (UTR) of the Drosophila bicoid (bcd) mRNA. Furthermore, covariations occurring in the helices of these conserved RNA structures are analyzed. Two physical parameters are found to be important determinants of the evolution of compensatory mutations: the length of a helix and the distance between base-pairing nucleotides. For the helices of bcd 3' UTR mRNA and RNase P RNA, a positive correlation between the rate of compensatory evolution and helix length is found. The analysis of Drosophila bcd 3' UTR mRNA further revealed that the rate of compensatory evolution decreases with the physical distance between base-pairing residues. This result is in qualitative agreement with Kimura's model of compensatory fitness interactions, which assumes that mutations occurring in RNA helices are individually deleterious but become neutral in appropriate combinations.

Analysis of Variance↗

Characterization of an RNA bulge structure by Fourier transform infrared spectroscopy.

There may be several advantages associated with an antisense oligonucleotide that induces a bulged structure into its RNA target molecule. Many structures of RNA bulges are elucidated from single-stranded RNA models. However, a two-component system is the minimum requirement for a realistic antisense model. We have used Fourier transform infrared spectroscopy to investigate a single-stranded RNA oligonucleotide with known NMR solution structure, constructed to model a five nucleotide bulge, and its two-component oligonucleotide counterpart. The infrared spectra show A-helical base-paired stems and non-base-paired loops in both systems. The nucleosides are mainly in an anti-conformation. Both N-type and S-type of sugar puckers can be inferred from the infrared region sensitive to sugar conformations. The S-type of sugar pucker is likely to be associated with the nucleotides in the bulge. The FTIR results display an overall structural similarity between the two model systems.

Base Sequence↗

Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure.

Human immunodeficiency virus type 1 Rev protein modulates the distribution of viral mRNAs from the nucleus to the cytoplasm by interaction with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To identify the minimal functional elements of RRE, we evaluated mutant RREs for Rev binding in vitro and Rev response in vivo in the context of a Gag expression plasmid. The critical functional elements fold into a structure composed of a stem-loop A, formed by the ends of the RRE, joined to a branched stem-loop B/B1/B2, between bases 49 and 113. The 5' 132 nucleotides of RRE, RREDDE, which possessed a similar structure, bound Rev efficiently but were nonfunctional in vivo, implying separate binding and functional domains within the RRE. Excision of stem-loop A reduced Rev binding significantly and abolished the in vivo Rev response. The B2 branch could be removed without severe impairment of binding, but deletions in the B1 branch significantly reduced binding and function. However, deletion of 12 nucleotides, including the 5' strand of stem B, abolished both binding and function, while excision of the 3' strand of stem B only reduced them. Maintenance of the native RRE secondary structure alone was not sufficient for Rev recognition. Many mutations that altered the primary structure of the critical region while preserving the original RNA conformation were Rev responsive. However, mutations that changed a 5'..CACUAUGGG..3' sequence in the B stem, without affecting the overall structure abolished both in vitro Rev binding and the in vivo Rev response.

Base Sequence↗

SCOR: a Structural Classification of RNA database.

The Structural Classification of RNA (SCOR) database provides a survey of the three-dimensional motifs contained in 259 NMR and X-ray RNA structures. In one classification, the structures are grouped according to function. The RNA motifs, including internal and external loops, are also organized in a hierarchical classification. The 259 database entries contain 223 internal and 203 external loops; 52 entries consist of fully complementary duplexes. A classification of the well-characterized tertiary interactions found in the larger RNA structures is also included along with examples. The SCOR database is accessible at http://scor.lbl.gov.

Animals↗

Identifying good predictions of RNA secondary structure.

Predicting the secondary structure of RNA molecules from the knowledge of the primary structure (the sequence of bases) is still a challenging task. There are algorithms that provide good results e.g. based on the search for an energetic optimal configuration. However the output of such algorithms does not always give the real folding of the molecule and therefore a feature to judge the reliability of the prediction would be appreciated. In this paper we present results on the expected structural behavior of LSU rRNA derived using a stochastic context-free grammar and generating functions. We show how these results can be used to judge the predictions made for LSU rRNA by any algorithm. In this way it will be possible to identify those predictions which are close to the natural folding of the molecule with a probability of 97% of success.

Algorithms↗

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids.

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.

DNA↗

[Prediction of the ensembles of RNA secondary structures. Kinetic analysis of self-organization].

A new approach to the problem of prediction of secondary structures of RNA, which is based on the kinetic analysis of self-organising molecules is proposed. Structural reconstructions that take place during formation of secondary structures are described in terms of Markov process. A set of states and probability transition were defined. Monte-Carlo methods were used to describe this process. Probability distributions of various secondary structures depending on time are given. Examples of calculations for ensembles of secondary structures of some tRNAs are described. An effective method of steady-state ensemble research, which is based on a quick RESETTING of all possible variance of the secondary structures of RNAs is given. By ascribing to each of these structures the value of probabilities as a function of free energy it was possible to obtain the Boltzmann ensemble of secondary structures.

Escherichia coli↗