Motivation: Protein synthesis is not a straight forward process and one gene locus can produce many isoforms, for example, by starting mRNA translation from alternative start sites. altORF evaluator (altORFev) predicts alternative open reading frames within eukaryotic mRNA translated by a linear scanning mechanism and its modifications (leaky scanning and reinitiation). The program reveals the efficiently translated altORFs recognized by the majority of 40S ribosomal subunits landing on the 5'-end of an mRNA. This information aids to reveal the functions of eukaryotic genes connected to synthesis of either unknown isoforms of annotated proteins or new unrelated polypeptides. Availability and Implementation: altORFev is available at http://www.bionet.nsc.ru/AUGWeb/ and has been developed in Java 1.8 using the BioJava library; and the Vaadin framework to produce the web service. Contact: [email protected].

BACKGROUND: The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties. RESULTS: We evaluated interdependency between the start codon context and mRNA secondary structure at the CDS beginning: it was found that a suboptimal start codon context significantly correlated with higher base pairing probabilities at positions 13 - 17 of CDS of human and mouse mRNAs. It is likely that the downstream hairpins are used to enhance translation of some mammalian mRNAs in vivo. Thus, we have developed a tool, AUG_hairpin, to predict local stem-loop structures located within the defined region at the beginning of mRNA coding part. The implemented algorithm is based on the available published experimental data on the CDS-located stem-loop structures influencing the recognition of upstream start codons. CONCLUSION: An occurrence of a potential secondary structure downstream of start AUG codon in a suboptimal context (or downstream of a potential non-AUG start codon) may provide researchers with a testable assumption on the presence of additional regulatory signal influencing mRNA translation initiation rate and the start codon choice. AUG_hairpin, which has a convenient Web-interface with adjustable parameters, will make such an evaluation easy and efficient.

MOTIVATION: The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. However, mRNAs with a suboptimal context of start AUG codon are relatively abundant. It is likely that at least some mRNAs with suboptimal start codon context contain the other signals providing additional information for efficient AUG recognition. RESULTS: Frequency of AUG codons at the beginning of the coding part of eukaryotic mRNAs was analyzed in relation to the context of translation start codon. It was found that the observed downstream AUG content in the mRNAs with optimal start codon context was close to the expected value, whereas it was significantly higher in the mRNAs with a suboptimal context. It is likely that downstream AUG codons can often be utilized as additional start sites to increase translation rate of mRNAs with a suboptimal context of the annotated start codon and many eukaryotic proteins can be characterized by some N-end heterogeneity.

MOTIVATION: According to scanning model, 40S ribosomal subunits can either initiate translation at start AUG codon in suboptimal context or miss it and initiate translation at downstream AUG(s), thereby producing several proteins. Functional significance of such a protein translational polymorphism is still unknown. RESULTS: We compared predicted subcellular localizations of annotated Arabidopsis thaliana proteins and their potential N-terminally truncated forms started from the nearest downstream in-frame AUG codons. It was found that localizations of full and N-truncated proteins differ in many cases: 12.2% of N-truncated proteins acquired sorting signals de novo and 5.7% changed their predicted subcellular locations (mitochodria, chloroplast or secretory pathway). It is likely that the in-frame downstream AUGs may be frequently utilized to synthesize proteins possessing new functional properties and such a translational polymorphism may serve as an important source of cellular and organelle proteomes.

MOTIVATION: The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. RESULTS: We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. AVAILABILITY: An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. CONTACT: [email protected], [email protected].

MOTIVATION: It is well known that eukaryotic mRNAs are translated at different levels depending on their sequence characteristics. Evaluation of mRNA translatability is of importance in prediction of the gene expression pattern by computer methods and to improve the recognition of mRNAs within cloned nucleotide sequences. It may also be used in biotechnological experiments to optimize the expression of foreign genes in transgenic organisms. RESULTS: The sets of 5' untranslated region characteristics, significantly different between mRNAs encoding abundant and scarce polypeptides, were determined for mammals, dicot plants and monocot plants, and collected in the LEADER_RNA database. Computer tools for the prediction of mRNA translatability are presented. AVAILABILITY: Programs for mRNA translatability prediction are available at http://wwwmgs.bionet.nsc. ru/programs/acts2/mo_mRNA.htm (for monocots), http://wwwmgs.bionet. nsc.ru/programs/acts2/di_mRNA.htm (for dicots) and http://wwwmgs. bionet.nsc.ru/programs/acts2/ma_mRNA.htm (for mammals). The LEADER_RNA database may be accessed at: http://wwwmgs.bionet.nsc. ru/systems/LeaderRNA/.

MOTIVATION: The goal of the work was to develop a WWW-oriented computer system providing a maximal integration of informational and software resources on the regulation of gene expression and navigation through them. Rapid growth of the variety and volume of information accumulated in the databases on regulation of gene expression necessarily requires the development of computer systems for automated discovery of the knowledge that can be further used for analysis of regulatory genomic sequences. RESULTS: The GeneExpress system developed includes the following major informational and software modules: (1) Transcription Regulation (TRRD) module, which contains the databases on transcription regulatory regions of eukaryotic genes and TRRD Viewer for data visualization; (2) Site Activity Prediction (ACTIVITY), the module for analysis of functional site activity and its prediction; (3) Site Recognition module, which comprises (a) B-DNA-VIDEO system for detecting the conformational and physicochemical properties of DNA sites significant for their recognition, (b) Consensus and Weight Matrices (ConsFrec) and (c) Transcription Factor Binding Sites Recognition (TFBSR) systems for detecting conservative contextual regions of functional sites and their recognition; (4) Gene Networks (GeneNet), which contains an object-oriented database accumulating the data on gene networks and signal transduction pathways, and the Java-based Viewer for exploration and visualization of the GeneNet information; (5) mRNA Translation (Leader mRNA), designed to analyze structural and contextual properties of mRNA 5'-untranslated regions (5'-UTRs) and predict their translation efficiency; (6) other program modules designed to study the structure-function organization of regulatory genomic sequences and regulatory proteins. AVAILABILITY: GeneExpress is available at http://wwwmgs.bionet.nsc. ru/systems/GeneExpress/ and the links to the mirror site(s) can be found at http://wwwmgs.bionet.nsc.ru/mgs/links/mirrors.html+ ++.