Connections between alternative transcription and alternative splicing in mammals.

The majority of mammalian genes produce multiple transcripts resulting from alternative splicing (AS) and/or alternative transcription initiation (ATI) and alternative transcription termination (ATT). Comparative analysis of the number of alternative nucleotides, isoforms, and introns per locus in genes with different types of alternative events suggests that ATI and ATT contribute to the diversity of human and mouse transcriptome even more than AS. There is a strong negative correlation between AS and ATI in 5' untranslated regions (UTRs) and AS in coding sequences (CDSs) but an even stronger positive correlation between AS in CDSs and ATT in 3' UTRs. These observations could reflect preferential regulation of distinct, large groups of genes by different mechanisms: 1) regulation at the level of transcription initiation and initiation of translation resulting from ATI and AS in 5' UTRs and 2) posttranslational regulation by different protein isoforms. The tight linkage between AS in CDSs and ATT in 3' UTRs suggests that variability of 3' UTRs mediates differential translational regulation of alternative protein forms. Together, the results imply coordinate evolution of AS and alternative transcription, processes that occur concomitantly within gene expression factories.

Optimization of duplex stability and terminal asymmetry for shRNA design.

Prediction of efficient oligonucleotides for RNA interference presents a serious challenge, especially for the development of genome-wide RNAi libraries which encounter difficulties and limitations due to ambiguities in the results and the requirement for significant computational resources. Here we present a fast and practical algorithm for shRNA design based on the thermodynamic parameters. In order to identify shRNA and siRNA features universally associated with high silencing efficiency, we analyzed structure-activity relationships in thousands of individual RNAi experiments from publicly available databases (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/). Using this statistical analysis, we found free energy ranges for the terminal duplex asymmetry and for fully paired duplex stability, such that shRNAs or siRNAs falling in both ranges have a high probability of being efficient. When combined, these two parameters yield a approximately 72% success rate on shRNAs from the siRecords database, with the target RNA levels reduced to below 20% of the control. Two other parameters correlate well with silencing efficiency: the stability of target RNA and the antisense strand secondary structure. Both parameters also correlate with the short RNA duplex stability; as a consequence, adding these parameters to our prediction scheme did not substantially improve classification accuracy. To test the validity of our predictions, we designed 83 shRNAs with optimal terminal asymmetry, and experimentally verified that small shifts in duplex stability strongly affected silencing efficiency. We showed that shRNAs with short fully paired stems could be successfully selected by optimizing only two parameters: terminal duplex asymmetry and duplex stability of the hypothetical cleavage product, which also relates to the specificity of mRNA target recognition. Our approach performs at the level of the best currently utilized algorithms that take into account prediction of the secondary structure of the target and antisense RNAs, but at significantly lower computational costs. Based on this study, we created the si-shRNA Selector program that predicts both highly efficient shRNAs and functional siRNAs (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/).

Distinct patterns of expression and evolution of intronless and intron-containing mammalian genes.

Comparison of expression levels and breadth and evolutionary rates of intronless and intron-containing mammalian genes shows that intronless genes are expressed at lower levels, tend to be tissue specific, and evolve significantly faster than spliced genes. By contrast, monomorphic spliced genes that are not subject to detectable alternative splicing and polymorphic alternatively spliced genes show similar statistically indistinguishable patterns of expression and evolution. Alternative splicing is most common in ancient genes, whereas intronless genes appear to have relatively recent origins. These results imply tight coupling between different stages of gene expression, in particular, transcription, splicing, and nucleocytosolic transport of transcripts, and suggest that formation of intronless genes is an important route of evolution of novel tissue-specific functions in animals.

Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158)met) position, designated as low (LPS), average (APS), and high pain sensitive (HPS), are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs), accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224) is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488) are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.

Computational models with thermodynamic and composition features improve siRNA design.

BACKGROUND: Small interfering RNAs (siRNAs) have become an important tool in cell and molecular biology. Reliable design of siRNA molecules is essential for the needs of large functional genomics projects. RESULTS: To improve the design of efficient siRNA molecules, we performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature. We used this training set to select siRNA features and optimize computational models. We identified 18 parameters that correlate significantly with silencing efficiency. Some of these parameters characterize only the siRNA sequence, while others involve the whole mRNA. Most importantly, we derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand. The position-dependent consensus is based on correlation and t-test analyses of the training set, and accounts for both significantly preferred and avoided nucleotides in all sequence positions. On the training set, the two parameters' correlation with silencing efficiency was 0.5 and 0.36, respectively. Among other features, a dinucleotide content index and the frequency of potential targets for siRNA in the mRNA added predictive power to our model (R = 0.55). We showed that our model is effective for predicting the efficiency of siRNAs at different concentrations. We optimized a neural network model on our training set using three parameters characterizing the siRNA sequence, and predicted efficiencies for the test siRNA dataset recently published by Novartis. On this validation set, the correlation coefficient between predicted and observed efficiency was 0.75. Using the same model, we performed a transcriptome-wide analysis of optimal siRNA targets for 22,600 human mRNAs. CONCLUSION: We demonstrated that the properties of the siRNAs themselves are essential for efficient RNA interference. The 5' ends of antisense strands of efficient siRNAs are U-rich and possess a content similarity to the pyrimidine-rich oligonucleotides interacting with the polypurine RNA tracks that are recognized by RNase H. The advantage of our method over similar methods is the small number of parameters. As a result, our method requires a much smaller training set to produce consistent results. Other mRNA features, though expensive to compute, can slightly improve our model.

Congruent evolution of different classes of non-coding DNA in prokaryotic genomes.

Prokaryotic genomes are considered to be 'wall-to-wall' genomes, which consist largely of genes for proteins and structural RNAs, with only a small fraction of the genomic DNA allotted to intergenic regions, which are thought to typically contain regulatory signals. The majority of bacterial and archaeal genomes contain 6-14% non-coding DNA. Significant positive correlations were detected between the fraction of non-coding DNA and inter- and intra-operonic distances, suggesting that different classes of non-coding DNA evolve congruently. In contrast, no correlation was found between any of these characteristics of non-coding sequences and the number of genes or genome size. Thus, the non-coding regions and the gene sets in prokaryotes seem to evolve in different regimes. The evolution of non-coding regions appears to be determined primarily by the selective pressure to minimize the amount of non-functional DNA, while maintaining essential regulatory signals, because of which the content of non-coding DNA in different genomes is relatively uniform and intra- and inter-operonic non-coding regions evolve congruently. In contrast, the gene set is optimized for the particular environmental niche of the given microbe, which results in the lack of correlation between the gene number and the characteristics of non-coding regions.

Purifying and directional selection in overlapping prokaryotic genes.

In overlapping genes, the same DNA sequence codes for two proteins using different reading frames. Analysis of overlapping genes can help in understanding the mode of evolution of a coding region from noncoding DNA. We identified 71 pairs of convergent genes, with overlapping 3' ends longer than 15 nucleotides, that are conserved in at least two prokaryotic genomes. Among the overlap regions, we observed a statistically significant bias towards the 123:132 phase (i.e. the second codon base in one gene facing the degenerate third position in the second gene). This phase ensures the least mutual constraint on nonconservative amino acid replacements in both overlapping coding sequences. The excess of this phase is compatible with directional (positive) selection acting on the overlapping coding regions. This could be a general evolutionary mode for genes emerging from noncoding sequences, in which the protein sequence has not been subject to selection.