Andrei A. Mironov

Assessing computational tools for the discovery of transcription factor binding sites

The prediction of regulatory elements is a problem where computational methods offer great hope. Over the past few years, numerous tools have become available for this task. The purpose of the current assessment is twofold: to provide some guidance to users regarding the accuracy of currently available tools in various settings, and to provide a benchmark of data sets for assessing future tools.

Comparative genomic analysis of T-box regulatory systems in bacteria

T-box antitermination is one of the main mechanisms of regulation of genes involved in amino acid metabolism in Gram-positive bacteria. T-box regulatory sites consist of conserved sequence and RNA secondary structure elements. Using a set of known T-box sites, we constructed the common pattern and used it to scan available bacterial genomes. New T-boxes were found in various Gram-positive bacteria, some Gram-negative bacteria (delta-proteobacteria), and some other bacterial groups (Deinococcales/Thermales, Chloroflexi, Dictyoglomi). The majority of T-box-regulated genes encode aminoacyl-tRNA synthetases. Two other groups of T-box-regulated genes are amino acid biosynthetic genes and transporters, as well as genes with unknown function. Analysis of candidate T-box sites resulted in new functional annotations. We assigned the amino acid specificity to a large number of candidate amino acid transporters and a possible function to amino acid biosynthesis genes. We then studied the evolution of the T-boxes. Analysis of the constructed phylogenetic trees demonstrated that in addition to the normal evolution consistent with the evolution of regulated genes, T-boxes may be duplicated, transferred to other genes, and change specificity. We observed several cases of recent T-box regulon expansion following the loss of a previously existing regulatory system, in particular, arginine regulon in Clostridium difficile and methionine regulon in Lactobacillaceae. Finally, we described a new structural class of T-boxes containing duplicated terminator-antiterminator elements and unusual reduced T-boxes regulating initiation of translation in the Actinobacteria.

Genome-Wide Molecular Clock and Horizontal Gene Transfer in Bacterial Evolution

We describe a simple theoretical framework for identifying orthologous sets of genes that deviate from a clock-like model of evolution. The approach used is based on comparing the evolutionary distances within a set of orthologs to a standard intergenomic distance, which was defined as the median of the distribution of the distances between all one-to-one orthologs. Under the clock-like model, the points on a plot of intergenic distances versus intergenomic distances are expected to fit a straight line. A statistical technique to identify significant deviations from the clock-like behavior is described. For several hundred analyzed orthologous sets representing three well-defined bacterial lineages, the alpha-Proteobacteria, the gamma-Proteobacteria, and the Bacillus-Clostridium group, the clock-like null hypothesis could not be rejected for approximately 70% of the sets, whereas the rest showed substantial anomalies. Subsequent detailed phylogenetic analysis of the genes with the strongest deviations indicated that over one-half of these genes probably underwent a distinct form of horizontal gene transfer, xenologous gene displacement, in which a gene is displaced by an ortholog from a different lineage. The remaining deviations from the clock-like model could be explained by lineage-specific acceleration of evolution. The results indicate that although xenologous gene displacement is a major force in bacterial evolution, a significant majority of orthologous gene sets in three major bacterial lineages evolved in accordance with the clock-like model. The approach described here allows rapid detection of deviations from this mode of evolution on the genome scale.

Starts of bacterial genes: estimating the reliability of computer predictions.

Exact mapping of gene starts is an important problem in the computer-assisted functional analysis of newly sequenced prokaryotic genomes. We describe an algorithm for finding ribosomal binding sites without a learning sample. This algorithm is particularly useful for analysis of genomes with little or no experimentally mapped genes. There is a clear correlation between the ribosomal binding site (RBS) properties of a given genome and the potential gene start prediction accuracy. This correlation is of considerable predictive power and may be useful for estimating the expected success of future genome analysis efforts. We also demonstrate that the RBS properties depend on the phylogenetic position of a genome.

RNAKinetics: a web server that models secondary structure kinetics of an elongating RNA.

The RNAKinetics server (http://www.ig-msk.ru/RNA/kinetics) is a web interface for the newly developed RNAKinetics software. The software models the dynamics of RNA secondary structure by the means of kinetic analysis of folding transitions of a growing RNA molecule. The result of the modeling is a kinetic ensemble, i.e. a collection of RNA structures that are endowed with probabilities, which depend on time. This approach gives comprehensive probabilistic description of RNA folding pathways, revealing important kinetic details that are not captured by the traditional structure prediction methods. The access to the RNAKinetics server is free.

Modulation of alternative splicing by long-range RNA structures in Drosophila

Accurate and efficient recognition of splice sites during pre-mRNA splicing is essential for proper transcriptome expression. Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances. Using phlyogenetic comparisons of intronic sequences among 12 Drosophila genomes, we elucidated a group of 202 highly conserved pairs of sequences, each at least nine nucleotides long, capable of forming stable stem structures. This set was highly enriched in alternatively spliced introns and introns with weak acceptor sites and long introns, and most occurred over long distances (>150 nucleotides). Experimentally, we analyzed the splicing of several of these introns using mini-genes in Drosophila S2 cells. Wild-type splicing patterns were changed by mutations that opened the stem structure, and restored by compensatory mutations that re-established the base-pairing potential, demonstrating that these secondary structures were indeed implicated in the splice site choice. Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns. Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

Positive Selection in Alternatively Spliced Exons of Human Genes

Alternative splicing is a well-recognized mechanism of accelerated genome evolution. We have studied single-nucleotide polymorphisms and human-chimpanzee divergence in the exons of 6672 alternatively spliced human genes, with the aim of understanding the forces driving the evolution of alternatively spliced sequences. Here, we show that alternatively spliced exons and exon fragments (alternative exons) from minor isoforms experience lower selective pressure at the amino acid level, accompanied by selection against synonymous sequence variation. The results of the McDonald-Kreitman test suggest that alternatively spliced exons, unlike exons constitutively included in the mRNA, are also subject to positive selection, with up to 27% of amino acids fixed by positive selection.

Base-calling algorithm with vocabulary (BCV) method for analyzing population sequencing chromatograms

Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3–14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing.

Evidence for widespread association of mammalian splicing and conserved long-range RNA structures

Pre-mRNA structure impacts many cellular processes, including splicing in genes associated with disease. The contemporary paradigm of RNA structure prediction is biased toward secondary structures that occur within short ranges of pre-mRNA, although long-range base-pairings are known to be at least as important. Recently, we developed an efficient method for detecting conserved RNA structures on the genome-wide scale, one that does not require multiple sequence alignments and works equally well for the detection of local and long-range base-pairings. Using an enhanced method that detects base-pairings at all possible combinations of splice sites within each gene, we report a list of RNA structures that could be involved in the regulation of splicing in mammals. We demonstrate statistically that there is a strong association between the occurrence of conserved RNA structures and alternative splicing, where local RNA structures are generally more frequent at alternative donor splice sites, while long-range structures are more associated with weak alternative acceptor splice sites. A fraction of the reported structures is associated with unannotated splicing events that are confirmed by RNA-seq data. As an example, we validated the RNA structure in the human SF1 gene using mini-genes in the HEK293 cell line. Point mutations that disrupted the base-pairing of two complementary boxes between exons 9 and 10 of this gene altered the splicing pattern, while the compensatory mutations that reestablished the base-pairing reverted splicing to that of the wild-type. There is statistical evidence for a Dscam-like class of mammalian genes, in which mutually exclusive RNA structures control mutually exclusive alternative splicing. In sum, we propose that long-range base-pairings carry an important, yet unconsidered part of the splicing code, and that, even by modest estimates, there must be thousands of such potentially regulatory structures conserved throughout the evolutionary history of mammals.

AN OVERVIEW OF THE ALGORITHMIC SESSION OF MCCMB'05

Genome analysis. This is a most classical bioinformatics topic that includes problems of local and global alignment of genomic sequences and related issues. Grigory Kucherov (LORIA, France) gave a talk on a general method for computing the sensitivity of seeds (small patterns used to spot potential local similarities), and more specifically “subset seeds”, a new concept of seeds. Inna Dubchak (Lawrence Berkeley National Lab, USA) described the VISTA system which is a software system that features several useful programs of genome alignment and analysis.