Aleksey Y. Ogurtsov

A periodic pattern of mRNA secondary structure created by the genetic code

Single-stranded mRNA molecules form secondary structures through complementary self-interactions. Several hypotheses have been proposed on the relationship between the nucleotide sequence, encoded amino acid sequence and mRNA secondary structure. We performed the first transcriptome-wide in silico analysis of the human and mouse mRNA foldings and found a pronounced periodic pattern of nucleotide involvement in mRNA secondary structure. We show that this pattern is created by the structure of the genetic code, and the dinucleotide relative abundances are important for the maintenance of mRNA secondary structure. Although synonymous codon usage contributes to this pattern, it is intrinsic to the structure of the genetic code and manifests itself even in the absence of synonymous codon usage bias at the 4-fold degenerate sites. While all codon sites are important for the maintenance of mRNA secondary structure, degeneracy of the code allows regulation of stability and periodicity of mRNA secondary structure. We demonstrate that the third degenerate codon sites contribute most strongly to mRNA stability. These results convincingly support the hypothesis that redundancies in the genetic code allow transcripts to satisfy requirements for both protein structure and RNA structure. Our data show that selection may be operating on synonymous codons to maintain a more stable and ordered mRNA secondary structure, which is likely to be important for transcript stability and translation. We also demonstrate that functional domains of the mRNA [5′-untranslated region (5′-UTR), CDS and 3′-UTR] preferentially fold onto themselves, while the start codon and stop codon regions are characterized by relaxed secondary structures, which may facilitate initiation and termination of translation.

Expansion of the Human μ-Opioid Receptor Gene Architecture: Novel Functional Variants

The μ-opioid receptor (OPRM1) is the principal receptor target for both endogenous and exogenous opioid analgesics. There are substantial individual differences in human responses to painful stimuli and to opiate drugs that are attributed to genetic variations in OPRM1. In searching for new functional variants, we employed comparative genome analysis and obtained evidence for the existence of an expanded human OPRM1 gene locus with new promoters, alternative exons and regulatory elements. Examination of polymorphisms within the human OPRM1 gene locus identified strong association between single nucleotide polymorphism (SNP) rs563649 and individual variations in pain perception. SNP rs563649 is located within a structurally conserved internal ribosome entry site (IRES) in the 5′-UTR of a novel exon 13-containing OPRM1 isoforms (MOR-1K) and affects both mRNA levels and translation efficiency of these variants. Furthermore, rs563649 exhibits very strong linkage disequilibrium throughout the entire OPRM1 gene locus and thus affects the functional contribution of the corresponding haplotype that includes other functional OPRM1 SNPs. Our results provide evidence for an essential role for MOR-1K isoforms in nociceptive signaling and suggest that genetic variations in alternative OPRM1 isoforms may contribute to individual differences in opiate responses.

Selective constraint in intergenic regions of human and mouse genomes

We aligned and analyzed 100 pairs of complete, orthologous intergenic regions from the human and mouse genomes (average length approximately 12 000 nucleotides). The alignments alternate between highly similar segments and dissimilar segments, indicating a wide variation of selective constraint. The average number of selectively constrained nucleotides within a mammalian intergenic region is at least 2000. This is threefold higher than within a nematode intergenic region and at least twofold higher than the number of selectively constrained nucleotides coding for an average protein. Because mammals possess only two- to threefold more proteins than Caenorhabditis elegans, the higher complexity of mammals might be primarily because of the functioning of intergenic DNA.

Comparison of approaches for rational siRNA design leading to a new efficient and transparent method

Current literature describes several methods for the design of efficient siRNAs with 19 perfectly matched base pairs and 2 nt overhangs. Using four independent databases totaling 3336 experimentally verified siRNAs, we compared how well several of these methods predict siRNA cleavage efficiency. According to receiver operating characteristics (ROC) and correlation analyses, the best programs were BioPredsi, ThermoComposition and DSIR. We also studied individual parameters that significantly and consistently correlated with siRNA efficacy in different databases. As a result of this work we developed a new method which utilizes linear regression fitting with local duplex stability, nucleotide position-dependent preferences and total G/C content of siRNA duplexes as input parameters. The new methods discrimination ability of efficient and inefficient siRNAs is comparable with that of the best methods identified, but its parameters are more obviously related to the mechanisms of siRNA action in comparison with BioPredsi. This permits insight to the underlying physical features and relative importance of the parameters. The new method of predicting siRNA efficiency is faster than that of ThermoComposition because it does not employ time-consuming RNA secondary structure calculations and has much less parameters than DSIR. It is available as a web tool called ‘siRNA scales’.

Positive selection at sites of multiple amino acid replacements since rat-mouse divergence.

New alleles become fixed owing to random drift of nearly neutral mutations or to positive selection of substantially advantageous mutations. After decades of debate, the fraction of fixations driven by selection remains uncertain. Within 9,390 genes, we analysed 28,196 codons at which rat and mouse differ from each other at two nucleotide sites and 1,982 codons with three differences. At codons where rat–mouse divergence involved two non-synonymous substitutions, both of them occurred in the same lineage, either rat or mouse, in 64% of cases; however, independent substitutions would occur in the same lineage with a probability of only 50%. All three non-synonymous substitutions occurred in the same lineage for 46% of codons, instead of the 25% expected. Furthermore, comparison of 12 pairs of prokaryotic genomes also shows clumping of multiple non-synonymous substitutions in the same lineage. This pattern cannot be explained by correlated mutation or episodes of relaxed negative selection, but instead indicates that positive selection acts at many sites of rapid, successive amino acid replacement.

OWEN: aligning long collinear regions of genomes

OWEN is an interactive tool for aligning two long DNA sequences that represents similarity between them by a chain of collinear local similarities. OWEN employs several methods for constructing and editing local similarities and for resolving conflicts between them. Alignments of sequences of lengths over 10(6) can often be produced in minutes. OWEN requires memory below 20 L, where L is the sum of lengths of the compared sequences.

Analysis of internal loops within the RNA secondary structure in almost quadratic time

MOTIVATION Evaluating all possible internal loops is one of the key steps in predicting the optimal secondary structure of an RNA molecule. The best algorithm available runs in time O(L(3)), L is the length of the RNA. RESULTS We propose a new algorithm for evaluating internal loops, its run-time is O(M(*)log(2)L), M < L(2) is a number of possible nucleotide pairings. We created a software tool Afold which predicts the optimal secondary structure of RNA molecules of lengths up to 28 000 nt, using a computer with 2 Gb RAM. We also propose algorithms constructing sets of conditionally optimal multi-branch loop free (MLF) structures, e.g. the set that for every possible pairing (x, y) contains an optimal MLF structure in which nucleotides x and y form a pair. All the algorithms have run-time O(M(*)log(2)L).

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 ∼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/).

Evolution at protein ends: major contribution of alternative transcription initiation and termination to the transcriptome and proteome diversity in mammals

Alternative splicing (AS), alternative transcription initiation (ATI) and alternative transcription termination (ATT) create the extraordinary complexity of transcriptomes and make key contributions to the structural and functional diversity of mammalian proteomes. Analysis of mammalian genomic and transcriptomic data shows that contrary to the traditional view, the joint contribution of ATI and ATT to the transcriptome and proteome diversity is quantitatively greater than the contribution of AS. Although the mean numbers of protein-coding constitutive and alternative nucleotides in gene loci are nearly identical, their distribution along the transcripts is highly non-uniform. On average, coding exons in the variable 5′ and 3′ transcript ends that are created by ATI and ATT contain approximately four times more alternative nucleotides than core protein-coding regions that diversify exclusively via AS. Short upstream exons that encompass alternative 5′-untranslated regions and N-termini of proteins evolve under strong nucleotide-level selection whereas in 3′-terminal exons that encode protein C-termini, protein-level selection is significantly stronger. The groups of genes that are subject to ATI and ATT show major differences in biological roles, expression and selection patterns.

RAId_DbS: Peptide Identification using Database Searches with Realistic Statistics

BackgroundThe key to mass-spectrometry-based proteomics is peptide identification. A major challenge in peptide identification is to obtain realistic E-values when assigning statistical significance to candidate peptides.ResultsUsing a simple scoring scheme, we propose a database search method with theoretically characterized statistics. Taking into account possible skewness in the random variable distribution and the effect of finite sampling, we provide a theoretical derivation for the tail of the score distribution. For every experimental spectrum examined, we collect the scores of peptides in the database, and find good agreement between the collected score statistics and our theoretical distribution. Using Students t-tests, we quantify the degree of agreement between the theoretical distribution and the score statistics collected. The T-tests may be used to measure the reliability of reported statistics. When combined with reported P-value for a peptide hit using a score distribution model, this new measure prevents exaggerated statistics. Another feature of RAId_DbS is its capability of detecting multiple co-eluted peptides. The peptide identification performance and statistical accuracy of RAId_DbS are assessed and compared with several other search tools. The executables and data related to RAId_DbS are freely available upon request.