Prediction of RNA secondary structure based on helical regions distribution

MOTIVATION: RNAs play an important role in many biological processes and knowing their structure is important in understanding their function. Due to difficulties in the experimental determination of RNA secondary structure, the methods of theoretical prediction for known sequences are often used. Although many different algorithms for such predictions have been developed, this problem has not yet been solved. It is thus necessary to develop new methods for predicting RNA secondary structure. The most-used at present is Zuker's algorithm which can be used to determine the minimum free energy secondary structure. However many RNA secondary structures verified by experiments are not consistent with the minimum free energy secondary structures. In order to solve this problem, a method used to search a group of secondary structures whose free energy is close to the global minimum free energy was developed by Zuker in 1989. When considering a group of secondary structures, if there is no experimental data, we cannot tell which one is better than the others. This case also occurs in combinatorial and heuristic methods. These two kinds of methods have several weaknesses. Here we show how the central limit theorem can be used to solve these problems. RESULTS: An algorithm for predicting RNA secondary structure based on helical regions distribution is presented, which can be used to find the most probable secondary structure for a given RNA sequence. It consists of three steps. First, list all possible helical regions. Second, according to central limit theorem, estimate the occurrence probability of every helical region based on the Monte Carlo simulation. Third, add the helical region with the biggest probability to the current structure and eliminate the helical regions incompatible with the current structure. The above processes can be repeated until no more helical regions can be added. Take the current structure as the final RNA secondary structure. In order to demonstrate the confidence of the program, a test on three RNA sequences: tRNAPhe, Pre-tRNATyr, and Tetrahymena ribosomal RNA intervening sequence, is performed. AVAILABILITY: The program is written in Turbo Pascal 7.0. The source code is available upon request. CONTACT: Wujj@nic.bmi.ac.cn or Liwj@mail.bmi.ac.cn     

Shaping space: the possible and the attainable in RNA genotype-phenotype mapping

Understanding which phenotypes are accessible from which genotypes is fundamental for understanding the evolutionary process. This notion of accessibility can be used to define a relation of nearness among phenotypes, independently of their similarity. Because of neutrality, phenotypes denote equivalence classes of genotypes. The definition of neighborhood relations among phenotypes relies, therefore, on the statistics of neighborhood relations among equivalence classes of genotypes in genotype space. The folding of RNA sequence (genotypes) into secondary structures (phenotypes) is an ideal case to implement these concepts. We study the extent to which the folding of RNA sequence induces a "statistical topology" on the set of minimum free energy secondary structures. The resulting nearness relation suggests a notion of "continuous" structure transformation. We can, then rationalize major transitions in evolutionary trajectories at the level of RNA structures by identifying those transformations which are irreducibly discontinuous. This is shown by means of computer simulations. The statistical topology organizing the set of RNA shapes explains why neutral drift in sequence space plays a key role in evolutionary optimization.     

Comparative structure analysis of vertebrate ribonuclease P RNA

Ribonuclease P cleaves 5'-precursor sequences from pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the protein component(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comparative studies of the structure of this RNA from diverse organisms, including eucaryotes, primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comparative studies. To expand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 partial RNase P RNA genes from 11 additional vertebrate taxa representing most extant major vertebrate lineages. Based on a recently proposed structure of the core elements of RNase P RNA, we aligned the sequences and propose a minimum consensus secondary structure for the vertebrate RNase P RNA.     

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

BACKGROUND: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. RESULTS: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. CONCLUSIONS: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

Assessing the reliability of RNA folding using statistical mechanics

We have analyzed the base-pairing probability distributions of 16 S and 16 S-like, and 23 S and 23 S-like ribosomal RNAs of Archaea, Bacteria, chloroplasts, mitochondria and Eukarya, as predicted by the partition function approach for RNA folding introduced by McCaskill. A quantitative analysis of the reliability of RNA folding is done by comparing the base-pairing probability distributions with the structures predicted by comparative sequence analysis (comparative structures). We distinguish two factors that show a relationship to the reliability of RNA minimum free energy structure. The first factor is the dominance of one particular base-pair or the absence of base-pairing for a given base within the base-pairing probability distribution (BPPD). We characterize the BPPD per base, including the probability of not base-pairing, by its Shannon entropy (S). The S value indicates the uncertainty about the base-pairing of a base: low S values result from BPPDs that are strongly dominated by a single base-pair or by the absence of base-pairing. We show that bases with low S values have a relatively high probability that their minimum free energy (MFE) structure corresponds to the comparative structure. The BPPDs of prokaryotes that live at high temperatures (thermophilic Archaea and Bacteria) have, calculated at 37 degrees C, lower S values than the BPPDs of prokaryotes that live at lower temperatures (mesophilic and psychrophilic Archaea and Bacteria). This reflects an adaptation of the ribosomal RNAs to the environmental temperature. A second factor that is important to consider with regard to the reliability of MFE structure folding is a variable degree of applicability of the thermodynamic model of RNA folding for different groups of RNAs. Here we show that among the bases that show low S values, the Archaea and Bacteria have similar, high probabilities (0.96 and 0.94 in 16 S and 0.93 and 0.91 in 23 S, respectively) that the MFE structure corresponds to the comparative structure. These probabilities are lower in the chloroplasts (16 S 0.91, 23 S 0.79), mitochondria (16 S-like 0.89, 23 S-like 0.69) and Eukarya (18 S 0.81, 28 S 0.86).     

Electron microscopic evidence for splicing of Moloney murine leukemia virus RNAs

Poly (A) containing RNA extracted from Moloney murine leukemia virus infected mouse cells was hybridized with long single-stranded complementary DNA, prepared in detergent disrupted virions. Visualization of the hybrids in the electron microscope revealed among the structures, circles and circles with tails. Measurements performed on the circular molecules revealed two major species with circumferences corresponding to 3 and 8.2 kilobases. The latter structures had identical size to circles obtained after annealing of cDNA with the viral genome, 35S RNA. Circularization of a small viral RNA (3 kb) from infected cells in the RNA-cDNA hybrids is a direct evidence that like the 35S RNA it shares similar nucleotide sequences at both the 5' and 3' ends. The presence of 5' end sequences common to the two RNA species indicates the existence of a spliced viral RNA. Furthermore, based on the circularization of viral RNA in the hybrids, we suggest a new way to quantitate and determine the lengths of spliced RNA in retrovirus infected cells.     

Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions

We explore the ability of a simple simulated annealing procedure to assemble native-like structures from fragments of unrelated protein structures with similar local sequences using Bayesian scoring functions. Environment and residue pair specific contributions to the scoring functions appear as the first two terms in a series expansion for the residue probability distributions in the protein database; the decoupling of the distance and environment dependencies of the distributions resolves the major problems with current database-derived scoring functions noted by Thomas and Dill. The simulated annealing procedure rapidly and frequently generates native-like structures for small helical proteins and better than random structures for small beta sheet containing proteins. Most of the simulated structures have native-like solvent accessibility and secondary structure patterns, and thus ensembles of these structures provide a particularly challenging set of decoys for evaluating scoring functions. We investigate the effects of multiple sequence information and different types of conformational constraints on the overall performance of the method, and the ability of a variety of recently developed scoring functions to recognize the native-like conformations in the ensembles of simulated structures.     

Evolutionary variation in bacterial RNase P RNAs

Sequences encoding RNase P RNAs from representatives of the last remaining classical phyla of Bacteria have been determined, completing a general phylogenetic survey of RNase P RNA sequence and structure. This broad sampling of RNase P RNAs allows some refinement of the secondary structure, and reveals patterns in the evolutionary variation of sequences and secondary structures. Although the sequences range from 100 to <25% identical to one another, and although only 40 of the nucleotides are invariant, there is considerable conservation of the underlying core of the RNA sequence. RNase P RNAs, like group I intron RNAs but unlike ribosomal RNAs, transfer RNAs or other highly conserved RNAs, are quite variable in secondary structure outside of this conserved structural core. Conservative regions of the RNA evolve by substitution of apparently interchangeable alternative structures, rather than the insertion and deletion of helical elements that occurs in the more variable regions of the RNA. In a remarkable case of convergent molecular evolution, most of the unusual structural elements of type B RNase P RNAs of the low G+C Gram-positive Bacteria have evolved independently in Thermomicrobium roseum , a member of the green non-sulfur Bacteria.     

Prediction of homology and divergence in the secondary structure of polypeptides

A quantitative procedure is described for the comparison of secondary structure of homologous proteins. Standard predictive methods are used to generate probability profiles from pairs of homologous amino acid sequences; correlation coefficients (R) are then computed between each pair of amino acids for alpha-helix (R alpha), extended structure (R beta), turn (R(t)), and coil (R(c)). R values are >0.2 for correctly aligned homologous sequences. Unrelated or incorrectly aligned sequences give R values near zero. Lack of correlation for a segment of otherwise well-correlated sequences is used to identify structural divergence, which is then evaluated graphically by using difference profiles. A combination of these techniques correctly predicts secondary structural differences between melittin or beta-endorphin and their respective synthetic analogs. The method is potentially useful to describe evolutionary changes in protein secondary structure as well as in the design of peptide analogs.     

Prediction of RNA Secondary Structure Based on Particle Swarm Optimization

A novel method for the prediction of RNA secondary structure was proposed based on the particle swarm optimization(PSO).PSO is known to be effective in solving many different types of optimization problems and known for being able to approximate the global optimal results in the solution space.We designed an efficient objective function according to the minimum free energy,the number of selected stems and the average length of selected stems.We calculated how many legal stems there were in the sequence,and selected some of them to obtain an optimal result using PSO in the right of the objective function.A method based on the improved particle swarm optimization(IPSO)was proposed to predict RNA secondary structure,which consisted of three stages.The first stage was applied to encoding the source sequences,and to exploring all the legal stems.Then,a set of encoded stems were created in order to prepare input data for the second stage.In the second stage,IPSO was responsible for structure selection.At last,the optimal result was obtained from the secondary structures selected via IPSO.Nine sequences from the comparative RNA website were selected for the evaluation of the proposed method.Compared with other six methods,the proposed method decreased the complexity and enhanced the sensitivity and specificity on the basis of the experiment results.     

A correlation between N2-dimethylguanosine presence and alternate tRNA conformers

Even though the evolutionary conservation of the cloverleaf model is strongly suggestive of powerful constraints on the secondary structure of functional tRNAs, some mitochondrial tRNAs cannot be folded into this form. From the optimal base pairing pattern of these recalcitrant tRNAs, structural correlations between the length of the anticodon stem and the lengths of connector regions between the two helical domains, formed by the coaxial stacking of the anticodon and D-stems and the acceptor and T-stems, have been derived and used to scan the tRNA and tRNA gene database. We show here that some cytosolic tRNA gene sequences that are compatible with the cloverleaf model can also be folded into patterns proposed for the unusual mitochondrial tRNAs. Furthermore, the ability to be folded into these atypical structures correlates in the mature RNA sequences with the presence of dimethylguanosine, whose role may be to prevent the unusual mitochondrial tRNA pattern folding.     

Size distribution of rat liver nuclear RNA containing mRNA sequences

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98% formamide. Complementary DNA (cDNA) was prepared from the 8--14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that: 1. All abundant 8--14-S poly(A)-containing mRNAs have larger nuclear precursor molecules; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length. 2. 70% of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S. 3. The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8--14-S mRNA.     

Automatic detection of conserved RNA structure elements in complete RNA virus genomes

We propose a new method for detecting conserved RNA secondary structures in a family of related RNA sequences. Our method is based on a combination of thermodynamic structure prediction and phylogenetic comparison. In contrast to purely phylogenetic methods, our algorithm can be used for small data sets of approximately 10 sequences, efficiently exploiting the information contained in the sequence variability. The procedure constructs a prediction only for those parts of sequences that are consistent with a single conserved structure. Our implementation produces reasonable consensus structures without user interference. As an example we have analysed the complete HIV-1 and hepatitis C virus (HCV) genomes as well as the small segment of hantavirus. Our method confirms the known structures in HIV-1 and predicts previously unknown conserved RNA secondary structures in HCV.     

RNA aptamers to the peptidyl transferase inhibitor chloramphenicol

BACKGROUND: The problem of how macromolecules adopt specific shapes to recognize small molecules in their environment is readily addressed through in vitro selections (the SELEX protocol). RNA-antibiotic interactions are particularly attractive systems for study because they provide an opportunity to expand our understanding of molecular recognition by RNA and to facilitate ribosomal modeling. Specifically, the antibiotic chloramphenicol (Cam) naturally binds bacterial ribosomes in the 'peptidyl transferase loop' of 23S ribosomal RNA to inhibit peptide bond formation. RESULTS: We identified Cam-binding RNA molecules ('aptamers') from two independent initial random RNA populations. Boundary determinations, ribonuclease S1 sensitivity analyses and the activity of truncated minimal RNAs identified a structural motif that is shared by sequences from both selections. The pseudosymmetric motif consists of a highly conserved central helix of five to six base pairs flanked by A-rich bulges and additional helices. Addition of Cam prior to ribonuclease S1 protected nucleotides in the conserved cores from cleavage. Reselection from a pool of mutated variants of the minimal aptamer further refined the sequence requirements for binding. Finally, we used proton nuclear magnetic resonance (NMR) to establish a 1:1 RNA: Cam stoichiometry of the complex. Both the protection and NMR data both show that Cam stabilizes the active fold of this aptamer. CONCLUSIONS: There are many different RNA sequences that can bind Cam. The Cam aptamers that we examined have a well-defined secondary structure with a binding pocket that appears to be stabilized by Cam. This RNA motif superficially resembles the Cam-binding site in 23S rRNA, although further work is needed to establish the significance of these similarities.     

Three-dimensional comparative modeling of RNA

Comparative sequence analysis and ERNA-3D software were used to model the three-dimensional structure of the small domain of signal recognition particle RNA. RNA secondary structures were established by allowing only phylogenetically-supported base pairs. The folding of the RNA molecules was constrained further to include a well-supported pseudoknot. Helical sections were oriented coaxially where a continuous helical stack was formed in the RNA of another species. Finally, RNA helices were placed at distances that preserved the connectivity of the molecule with the smallest number of single-stranded nucleotide residues as identified from the aligned sequences. We show that the comparative three-dimensional structure modeling approach is an extremely powerful tool as it requires only a critical number of carefully aligned sequences.     

Derivation of the three-dimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis

The secondary structure of bacterial RNase P RNA, a ribozyme responsible for the maturation of the 5' end of tRNAs, is well established on the basis of sequence comparison analysis. RNase P RNA secondary structures fall into two types, A and B, which share a common core formed by the assembly of two main folding domains, but differ in their peripheral elements.A revised alignment of 137 available sequences reveals new covariations allowing for the refinement of both types of secondary structures. Phylogenetic evidence is thus provided for the extension of stems P11, P14, P19, P10.1 and P15.1 through further canonical base-pairs or GAellipsisGA mismatches. These refinements led in turn to a new organization of the catalytic core, with coaxial stackings of helices P2 and P19 as well as P1 and P4. New inter-domain tertiary interactions involve loop L9 and helix P1 and loop L8 with helix P4. These features were incorporated into atomic-scale 3D models of RNase P RNA for representatives of each structural type, namely Escherichia coli and Bacillus subtilis. In each model, the juxtaposition of the core helices creates a cradle onto which the pre-tRNA substrate binds with most evolutionarily conserved residues converging towards the cleavage site. The inner cores of both types are stabilized similarly, albeit by different peripheral elements, emphasizing the modular and hierarchical organisation of the architecture of RNase P RNAs. Similarities are thus apparent between the type A modules, P16/P17/P6 and P13/P14, and their type B analogs, P5.1/P15.1 and P10. 1/P10.1a, respectively. Other noteworthy features of these models include compactness and good agreement with published crosslinking data.     

Triplet repeat instability and DNA topology: an expansion model based on statistical mechanics

The variance of writhe, the contribution of writhe to supercoiling, and the free energies of supercoiling were calculated for (CTG.CAG)n and (CGG.CCG)n triplet repeat sequences (TRS) by statistical mechanics from the bending and torsional moduli previously determined. Expansions of these sequences are inherited by non-mendelian transmission and are linked with several hereditary neuromuscular diseases. The variance of writhe was greater for the TRS than for random B-DNA. For random B-DNA, (CGG)n, and (CTG)n, the contribution of writhe to supercoiling was 70, 78, and 79%, whereas the free energy of supercoiling at a length of 10 kilobase pairs was 1040.RT, 760.RT, and 685.RT, respectively. These data indicate that the TRS are preferential sites for the partitioning of supercoiling. Calculations of the differences in free energy of supercoiling between the TRS and random B-DNA revealed a local minimum at approximately 520 base pairs. Human medical genetic studies have shown that individuals carrying up to 180-200 copies of TRS (540-600 base pairs, premutations) in the fragile X or myotonic dystrophy gene loci are usually asymptomatic, whereas large expansions (>200 repeats, full mutations), which lead to disease, are observed in their offspring. Therefore, the length corresponding to the local minimum in free energy of supercoiling correlates with the genetic breakpoint between premutation and full mutation. We propose that (a) TRS instability is mediated by DNA mispairing caused by the accumulation of supercoiling within the repeats, and (b) the expansions that take place at the premutation to full mutation threshold are associated with increased mispairing caused by the optimal partitioning of writhe within the TRS at this length.     

Rational and combinatorial strategies for designing oligonucleotides targeted to RNA structures

Many RNA structures play a key role in the regulation of gene expression. We designed synthetic oligonucleotides able to interact with folded RNA regions (see Toulmé et al., Biochimie (1996) 78, 663-673, for a review). We have demonstrated that a decanucleotide can form a triple helix with the stem of the hairpin responsible for ribosomal frame-shifting of the gag-pro message of HTLV-I, leading to the inhibition of translation. We have isolated, through an in vitro selection procedure, from a library composed of oligonucleotides with a random part of 30 nucleotides, sequences able to bind to the TAR RNA element of HIV-1 with a dissociation constant of 20-50 nM. The association between the two partners involve non-canonical interactions. This extends the range of potential targets for antisense sequences to functional RNA structures.