Computational RNA secondary structure design: empirical complexity and improved methods

Background  

We investigate the empirical complexity of the RNA secondary structure design problem, that is, the scaling of the typical difficulty of the design task for various classes of RNA structures as the size of the target structure is increased. The purpose of this work is to understand better the factors that make RNA structures hard to design for existing, high-performance algorithms. Such understanding provides the basis for improving the performance of one of the best algorithms for this problem, RNA-SSD, and for characterising its limitations.     

Translation and messenger RNA secondary structure

The possibility of translation being influenced by the messenger RNA secondary structure is investigated with the aid of a stochastic model. Simulations indicate that, at least for certain mRNA's, the mean ribosomal passage time decreases as the mean number of ribosomes on the messenger is increased. Furthermore, large variations in the passage times are found, in accordance with recent experimental results.     

RNA secondary structure and compensatory evolution

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.     

Vienna RNA secondary structure server

The Vienna RNA secondary structure server provides a web interface to the most frequently used functions of the Vienna RNA software package for the analysis of RNA secondary structures. It currently offers prediction of secondary structure from a single sequence, prediction of the consensus secondary structure for a set of aligned sequences and the design of sequences that will fold into a predefined structure. All three services can be accessed via the Vienna RNA web server at http://rna.tbi.univie.ac.at/.     

An RNA secondary structure workbench

A multiple approach to the study of RNA secondary structure is described which provides for the independent drawing of structures using base-pairing lists, for the generation of local structures in the form of hairpins, and for the generation of global structures by both Monte Carlo and dynamic programming methodologies. User-adjustable parameters provide for limiting the size of hairpin loops, bulges and inner loops, and constraints can be imposed relative to position-dependent base pairing.     

RNAmute: RNA secondary structure mutation analysis tool

Background  

RNAMute is an interactive Java application that calculates the secondary structure of all single point mutations, given an RNA sequence, and organizes them into categories according to their similarity with respect to the wild type predicted structure. The secondary structure predictions are performed using the Vienna RNA package. Several alternatives are used for the categorization of single point mutations: Vienna's RNAdistance based on dot-bracket representation, as well as tree edit distance and second eigenvalue of the Laplacian matrix based on Shapiro's coarse grain tree graph representation.     

General combinatorics of RNA secondary structure

The total number of RNA secondary structures of a given length with minimal hairpin loop length m(m>0) and with minimal stack length l(l>0) is computed, under the assumption that all base pairs can occur. Asymptotics are derived from the determination of recurrence relations of decomposition properties.     

Secondary structure of nuclear precursors of the informational RNA (pre-mRNA)]

The secondary structure of pre-mRNA species from mouse Ehrlich ascites carcinoma cells extracted with phenol at the temperatures either 55-65 degrees C or 65-85 degrees C was investigated. A fraction of the double helical regions of pre-mRNA was estimated by two methods: a) by recording of melting curves; b) by measuring fluorescence lifetimes of acridine orange dye adsorbed on the nucleic acid. This fraction was about 64-68%. Further lowering of ionic strength down to 0.024 results in 10-15% decrease in the fraction of double regions. Both lowering of ionic strength and increase of the temperature up to 50-55 degrees C results in despiralisation of pre-mRNA regions which contain more than 70% of AU-nucleotide pairs. Only regions containing mainly GC-nucleotide pairs remain double-stranded under heating to temperatures above 50 degrees C. These facts were established on the basis of studies of acriflavin dye complexes with pre-mRNA.     

RNAstructure: software for RNA secondary structure prediction and analysis

Background  

To understand an RNA sequence's mechanism of action, the structure must be known. Furthermore, target RNA structure is an important consideration in the design of small interfering RNAs and antisense DNA oligonucleotides. RNA secondary structure prediction, using thermodynamics, can be used to develop hypotheses about the structure of an RNA sequence.     

Revolutions in RNA secondary structure prediction

RNA structure formation is hierarchical and, therefore, secondary structure, the sum of canonical base-pairs, can generally be predicted without knowledge of the three-dimensional structure. Secondary structure prediction algorithms evolved from predicting a single, lowest free energy structure to their current state where statistics can be determined from the thermodynamic ensemble. This article reviews the free energy minimization technique and the salient revolutions in the dynamic programming algorithm methods for secondary structure prediction. Emphasis is placed on highlighting the recently developed method, which statistically samples structures from the complete Boltzmann ensemble.     

The kink-turn: a new RNA secondary structure motif

Analysis of the Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the kink-turn, or K-turn. The six K-turns in H.marismortui 23S rRNA superimpose with an r.m.s.d. of 1.7 A. There are two K-turns in the structure of Thermus thermophilus 16S rRNA, and the structures of U4 snRNA and L30e mRNA fragments form K-turns. The structure has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix. Its asymmetric internal loop is flanked by C-G base pairs on one side and sheared G-A base pairs on the other, with an A-minor interaction between these two helical stems. A derived consensus secondary structure for the K-turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5'-UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP. Five K-turns in 23S rRNA interact with nine proteins. While the observed K-turns interact with proteins of unrelated structures in different ways, they interact with L7Ae and two homologous proteins in the same way.     

CONTRAfold: RNA secondary structure prediction without physics-based models

MOTIVATION: For several decades, free energy minimization methods have been the dominant strategy for single sequence RNA secondary structure prediction. More recently, stochastic context-free grammars (SCFGs) have emerged as an alternative probabilistic methodology for modeling RNA structure. Unlike physics-based methods, which rely on thousands of experimentally-measured thermodynamic parameters, SCFGs use fully-automated statistical learning algorithms to derive model parameters. Despite this advantage, however, probabilistic methods have not replaced free energy minimization methods as the tool of choice for secondary structure prediction, as the accuracies of the best current SCFGs have yet to match those of the best physics-based models. RESULTS: In this paper, we present CONTRAfold, a novel secondary structure prediction method based on conditional log-linear models (CLLMs), a flexible class of probabilistic models which generalize upon SCFGs by using discriminative training and feature-rich scoring. In a series of cross-validation experiments, we show that grammar-based secondary structure prediction methods formulated as CLLMs consistently outperform their SCFG analogs. Furthermore, CONTRAfold, a CLLM incorporating most of the features found in typical thermodynamic models, achieves the highest single sequence prediction accuracies to date, outperforming currently available probabilistic and physics-based techniques. Our result thus closes the gap between probabilistic and thermodynamic models, demonstrating that statistical learning procedures provide an effective alternative to empirical measurement of thermodynamic parameters for RNA secondary structure prediction. AVAILABILITY: Source code for CONTRAfold is available at http://contra.stanford.edu/contrafold/.     

ProbKnot: Fast prediction of RNA secondary structure including pseudoknots

It is a significant challenge to predict RNA secondary structures including pseudoknots. Here, a new algorithm capable of predicting pseudoknots of any topology, ProbKnot, is reported. ProbKnot assembles maximum expected accuracy structures from computed base-pairing probabilities in O(N²) time, where N is the length of the sequence. The performance of ProbKnot was measured by comparing predicted structures with known structures for a large database of RNA sequences with fewer than 700 nucleotides. The percentage of known pairs correctly predicted was 69.3%. Additionally, the percentage of predicted pairs in the known structure was 61.3%. This performance is the highest of four tested algorithms that are capable of pseudoknot prediction. The program is available for download at: http://rna.urmc.rochester.edu/RNAstructure.html.     

Circles: automating the comparative analysis of RNA secondary structure

SUMMARY: Circles is a program for inferring RNA secondary structure using maximum weight matching. The program can read in an alignment in FASTA, ClustalW, or NEXUS format, compute a maximum weight matching, and export one or more secondary structures in various file formats. AVAILABILITY: The program is available at no cost from http://taxonomy.zoology.gla.ac.uk/rod/circles/ and requires Windows 95/98/NT. CONTACT: r.page@bio.gla.ac.uk     

RNA editing: complexity and complications

RNA editing in Trypanosomatids creates functional mitochondrial mRNAs by extensive uridylate (U) insertion and deletion as specified by small guide RNAs (gRNAs). Editing is catalysed by the multiprotein editosome. Over 20 of its protein components have been identified and additional proteins are likely to function in editing and its regulation. The functions of only a few editosome proteins have been determined. Surprisingly, there are related pairs or sets of editosome proteins, and insertion and deletion editing appear to be functionally and perhaps spatially separate. A model for the editosome is proposed, which has a catalysis domain with separate sectors for insertion and deletion editing. It also contains domains for anchor duplex and upstream RNA binding, which position the sequence to be edited in the catalysis domain.