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Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 46k miSS with sufficient read support, 9k (20%) are significantly expressed above the expected noise level, and among them 2.5k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP), tissue-specific expression of miSS, and miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. The analysis of human primary cell transcriptomes suggested that both tissue-specific and cell-type-specific factors contribute to the regulation of miSS expression. More than 20% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 15% of maSS and not more than 54% of tissue-specific miSS are noisy, while the proportion of noisy splice sites among non-significantly expressed miSS is above 63%., Author summary Pre-mRNA splicing is an important step in the processing of the genomic information during gene expression. During splicing, introns are excised from a gene transcript, and the remaining exons are ligated. Our work concerns one its particular subtype, which involves the so-called tandem alternative splice sites, a group of closely located exon borders that are used alternatively. We analyzed RNA-seq measurements of gene expression provided by the Genotype-Tissue Expression (GTEx) project, the largest to-date collection of such measurements in healthy human tissues, and constructed a detailed catalogue of tandem alternative splice sites. Within this catalogue, we characterized patterns of tissue-specific expression, regulation, impact on protein structure, and evolutionary selection acting on tandem alternative splice sites. In a number of genes, we predicted regulatory mechanisms that could be responsible for choosing one of many tandem alternative splice sites. The results of this study provide an invaluable resource for molecular biologists studying alternative splicing.

Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1’s roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.

Alternative splicing plays a major role in regulating the functional repertoire of the proteome. However, isoform-specific effects to protein-protein interactions (PPIs) are usually overlooked, making it impossible to judge the functional role of individual exons on a systems biology level. We overcome this barrier by integrating protein-protein interactions, domain-domain interactions and residue-level interactions information to lift exon expression analysis to a network level. Our user-friendly database DIGGER is available at https://exbio.wzw.tum.de/digger and allows users to seamlessly switch between isoform and exon-centric views of the interactome and to extract sub-networks of relevant isoforms, making it an essential resource for studying mechanistic consequences of alternative splicing.

Hepatitis C virus (HCV) causes a major health burden and can be effectively treated by direct-acting antivirals (DAAs). The non-structural protein 5A (NS5A), which plays a role in the viral genome replication, is one of the DAAs&rsquo, targets. Resistance-associated viruses (RAVs) harbouring NS5A resistance-associated mutations (RAMs) have been described at baseline and after therapy failure. A mutation from glutamine to arginine at position 30 (Q30R) is a characteristic RAM for the HCV sub/genotype (GT) 1a, but arginine corresponds to the wild type in the GT-1b, still, GT-1b strains are susceptible to NS5A-inhibitors. In this study, we show that GT-1b strains with R30Q often display other specific NS5A substitutions, particularly in positions 24 and 34. We demonstrate that in GT-1b secondary substitutions usually happen after initial R30Q development in the phylogeny, and that the chemical properties of the corresponding amino acids serve to restore the positive charge in this region, acting as compensatory mutations. These findings may have implications for RAVs treatment.

The origin and evolution of viruses is a subject of ongoing debate. In this study, we provide a full account of the evolutionary relationships between proteins of significant sequence and structural similarity found in viruses that belong to different classes according to the Baltimore classification. We show that such proteins can be found in viruses from all Baltimore classes. For protein families that include these proteins, we observe two patterns of the taxonomic spread. In the first pattern, they can be found in a large number of viruses from all implicated Baltimore classes. In the other pattern, the instances of the corresponding protein in species from each Baltimore class are restricted to a few compact clades. Proteins with the first pattern of distribution are products of so-called viral hallmark genes reported previously. Additionally, this pattern is displayed by the envelope glycoproteins from Flaviviridae and Bunyaviridae and helicases of superfamilies 1 and 2 that have homologs in cellular organisms. The second pattern can often be explained by horizontal gene transfer from the host or between viruses, an example being Orthomyxoviridae and Coronaviridae hemagglutinin esterases. Another facet of horizontal gene transfer comprises multiple independent introduction events of genes from cellular organisms into otherwise unrelated viruses.

Modern data on hepatitis C virus genome and polyprotein organization are summarized in this review. The functional roles of structural and nonstructural virus proteins, including their interaction with cellular regulatory proteins and structural cell elements, are described. Special features of the life cycle of the hepatitis C virus (that supplement our knowledge of viral hepatitis C pathogenesis) are presented.

Primary liver cancer (PLC) is a major public health concern worldwide, ranking third among the causes of death from cancer. Molecular pathogenesis of PLC is known to be especially sensitive to ethno-environmental variations that modulate mutation spectra in tumours. Despite a high prevalence of chronic liver diseases, the molecular epidemiology of PLC is still poorly known in Russia. To characterize the major genetic features of liver tumours in Russian populations, we conducted a pilot study on 34 PLC cases (28 hepatocellular, two cholangiocellular, and four mixed cases) among patients attending the Radiology and Roentgenology Hospital in Saint Petersburg. Point mutations were searched in 9 genes that are commonly altered in PLC, viz. TP53, CTNNB1, AXIN1, H/K/N-RAS, BRAF, PTEN, and NFE2L2. The genes TP53 and AXIN1 were mutated in 16% and 10% of the cases, respectively, whereas mutations of β-catenin were present in only 7% of samples, an unusual situation for Europe but common in East Asia. No R249S mutation indicative of exposure to aflatoxin B1 was detected in TP53. A single case harboured an NFE2L2 mutation. The loss of chromosome 18q was associated with early onset of tumours (mean 50 vs 62yrs, p=0.0252) and with the patient's place of birth in Caucasus or Siberia. A lack of any risk factor was noted in 47% of the patients, whereas only 23% of the patients were infected either by hepatitis virus B or C. An extension of the present cohort as well as further molecular studies are now warranted in order to understand the processes governing liver carcinogenesis affecting Russian populations.

The next generation sequencing technologies produce unprecedented amounts of data on the genetic sequence of individual organisms. These sequences carry a substantial amount of variation that may or may be not related to a phenotype. Phenotypically important part of this variation often comes in form of protein-sequence altering (non-synonymous) single nucleotide variants (nsSNVs). Here we present StructMAn, a Web-based tool for annotation of human and non-human nsSNVs in the structural context. StructMAn analyzes the spatial location of the amino acid residue corresponding to nsSNVs in the three-dimensional (3D) protein structure relative to other proteins, nucleic acids and low molecular-weight ligands. We make use of all experimentally available 3D structures of query proteins, and also, unlike other tools in the field, of structures of proteins with detectable sequence identity to them. This allows us to provide a structural context for around 20% of all nsSNVs in a typical human sequencing sample, for up to 60% of nsSNVs in genes related to human diseases and for around 35% of nsSNVs in a typical bacterial sample. Each nsSNV can be visualized and inspected by the user in the corresponding 3D structure of a protein or protein complex. The StructMAn server is available at http://structman.mpi-inf.mpg.de.

Progress in structure determination methods means that the set of experimentally determined 3D structures of proteins in complex with small molecules is growing exponentially. ProtChemSI exploits and extends this useful set of structures by both collecting and annotating the existing data as well as providing models of potential complexes inferred by protein or chemical structure similarity. The database currently includes 7704 proteins from 1803 organisms, 11,324 chemical compounds and 202, 289 complexes including 178,974 predicted. It is publicly available at http://pcidb.russelllab.org.

The currently available body of decoded amino acid sequences of various proteins exceeds manifold the experimental capabilities of their functional annotation. Therefore, in silico annotation using bioinformatics methods becomes increasingly important. Such annotation is actually a prediction; however, this can be an important starting point for further laboratory research. This work describes a new method for predicting functionally important protein sites, SDPsite, on the basis of identification of specificity determinants. The algorithm proposed utilizes a protein family aglinment and a phylogenetic tree to predict the conserved positions and specificity determinants, map them onto the protein structure, and search for clusters of the predicted positions. Comparison of the resulting predictions with experimental data and published predictions of functional sites by other methods demonstrates that the results of SDPsite agree well with experimental data and exceed the results obtained with the majority of previous methods. SDPsite is publicly available at http://bioinf.fbb.msu.ru/SDPsite .

Termination of protein synthesis is promoted in ribosomes by proper stop codon discrimination by class 1 polypeptide release factors (RFs). A large set of prokaryotic RFs differing in stop codon specificity, RF1 for UAG and UAA, and RF2 for UGA and UAA, was analyzed by means of a recently developed computational method allowing identification of the specificity-determining positions (SDPs) in families composed of proteins with similar but not identical function. Fifteen SDPs were identified within the RF1/2 superdomain II/IV known to be implicated in stop codon decoding. Three of these SDPs had particularly high scores. Five residues invariant for RF1 and RF2 [invariant amino acid residues (IRs)] were spatially clustered with the highest-scoring SDPs that in turn were located in two zones within the SDP/IR area. Zone 1 (domain II) included PxT and SPF motifs identified earlier by others as 'discriminator tripeptides'. We suggest that IRs in this zone take part in the recognition of U, the first base of all stop codons. Zone 2 (domain IV) possessed two SDPs with the highest scores not identified earlier. Presumably, they also take part in stop codon binding and discrimination. Elucidation of potential functional role(s) of the newly identified SDP/IR zones requires further experiments.

SDPpred (Specificity Determining Position prediction) is a tool for prediction of residues in protein sequences that determine the proteins' functional specificity. It is designed for analysis of protein families whose members have biochemically similar but not identical interaction partners (e.g. different substrates for a family of transporters). SDPpred predicts residues that could be responsible for the proteins' choice of their correct interaction partners. The input of SDPpred is a multiple alignment of a protein family divided into a number of specificity groups, within which the interaction partner is believed to be the same. SDPpred does not require information about the secondary or three-dimensional structure of proteins. It produces a set of the alignment positions (specificity determining positions) that determine differences in functional specificity. SDPpred is available at http://math.genebee.msu.ru/~psn/.

The increasing volume of genomic data opens new possibilities for analysis of protein function. We introduce a method for automated selection of residues that determine the functional specificity of proteins with a common general function (the specificity-determining positions [SDP] prediction method). Such residues are assumed to be conserved within groups of orthologs (that may be assumed to have the same specificity) and to vary between paralogs. Thus, considering a multiple sequence alignment of a protein family divided into orthologous groups, one can select positions where the distribution of amino acids correlates with this division. Unlike previously published techniques, the introduced method directly takes into account nonuniformity of amino acid substitution frequencies. In addition, it does not require setting arbitrary thresholds. Instead, a formal procedure for threshold selection using the Bernoulli estimator is implemented. We tested the SDP prediction method on the LacI family of bacterial transcription factors and a sample of bacterial water and glycerol transporters belonging to the major intrinsic protein (MIP) family. In both cases, the comparison with available experimental and structural data strongly supported our predictions.

Next-generation sequencing enables simultaneous analysis of hundreds of human genomes associated with a particular phenotype, for example, a disease. These genomes naturally contain a lot of sequence variation that ranges from single-nucleotide variants (SNVs) to large-scale structural rearrangements. In order to establish a functional connection between genotype and disease-associated phenotypes, one needs to distinguish disease drivers from neutral passenger variants. Functional annotation based on experimental assays is feasible only for a limited number of candidate mutations. Thus alternative computational tools are needed. A possible approach to annotating mutations functionally is to consider their spatial location relative to functionally relevant sites in three-dimensional (3D) structures of the harboring proteins. This is impeded by the lack of available protein 3D structures. Complementing experimentally resolved structures with reliable computational models is an attractive alternative. We developed a structure-based approach to characterizing comprehensive sets of non-synonymous single-nucleotide variants (nsSNVs): associated with cancer, non-cancer diseases and putatively functionally neutral. We searched experimentally resolved protein 3D structures for potential homology-modeling templates for proteins harboring corresponding mutations. We found such templates for all proteins with disease-associated nsSNVs, and 51 and 66% of proteins carrying common polymorphisms and annotated benign variants. Many mutations caused by nsSNVs can be found in protein–protein, protein–nucleic acid or protein–ligand complexes. Correction for the number of available templates per protein reveals that protein–protein interaction interfaces are not enriched in either cancer nsSNVs, or nsSNVs associated with non-cancer diseases. Whereas cancer-associated mutations are enriched in DNA-binding proteins, they are rarely located directly in DNA-interacting interfaces. In contrast, mutations associated with non-cancer diseases are in general rare in DNA-binding proteins, but enriched in DNA-interacting interfaces in these proteins. All disease-associated nsSNVs are overrepresented in ligand-binding pockets, and nsSNVs associated with non-cancer diseases are additionally enriched in protein core, where they probably affect overall protein stability.

Pairs of amino acid positions that evolve in a correlated manner are proposed to play important roles in protein structure or function. Methods to detect them might fare better with families for which sequences of thousands of closely related homologs are available than families with only a few distant relatives. We applied co-evolution analysis to thousands of sequences of HIV Gag, finding that the most significantly co-evolving positions are proximal in the quaternary structures of the viral capsid. A reduction in infectivity caused by mutating one member of a significant pair could be rescued by a compensatory mutation of the other.

International audience; The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99-124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of antit-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection.

Background Heavy metal resistance (HMR) in Eubacteria is regulated by a variety of systems including transcription factors from the MerR family (COG0789). The HMR systems are characterized by the complex signal structure (strong palindrome within a 19 or 20 bp promoter spacer), and usually consist of transporter and regulator genes. Some HMR regulons also include detoxification systems. The number of sequenced bacterial genomes is constantly increasing and even though HMR resistance regulons of the COG0789 type usually consist of few genes per genome, the computational analysis may contribute to the understanding of the cellular systems of metal detoxification. Results We studied the mercury (MerR), copper (CueR and HmrR), cadmium (CadR), lead (PbrR), and zinc (ZntR) resistance systems and demonstrated that combining protein sequence analysis and analysis of DNA regulatory signals it was possible to distinguish metal-dependent members of COG0789, assign specificity towards particular metals to uncharacterized loci, and find new genes involved in the metal resistance, in particular, multicopper oxidase and copper chaperones, candidate cytochromes from the copper regulon, new cadmium transporters and, possibly, glutathione-S-transferases. Conclusion Our data indicate that the specificity of the COG0789 systems can be determined combining phylogenetic analysis and identification of DNA regulatory sites. Taking into account signal structure, we can adequately identify genes that are activated using the DNA bending-unbending mechanism. In the case of regulon members that do not reside in single loci, analysis of potential regulatory sites could be crucial for the correct annotation and prediction of the specificity.

Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5′ untranslated region (5′UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5′UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the city's population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1_2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.

We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.

Biological networks are powerful tools for predicting undocumented relationships between molecules. The underlying principle is that existing interactions between molecules can be used to predict new interactions. Here we use this principle to suggest new protein-chemical interactions via the network derived from three-dimensional structures. For pairs of proteins sharing a common ligand, we use protein and chemical superimpositions combined with fast structural compatibility screens to predict whether additional compounds bound by one protein would bind the other. The method reproduces 84% of complexes in a benchmark, and we make many predictions that would not be possible using conventional modeling techniques. Within 19,578 novel predicted interactions are 7,793 involving 718 drugs, including filaminast, coumarin, alitretonin and erlotinib. The growth rate of confident predictions is twice that of experimental complexes, meaning that a complete structural drug-protein repertoire will be available at least ten years earlier than by X-ray and NMR techniques alone., Author Summary Predicting drug-target interactions is a hot topic, and many efforts have been undertaken to do this, many using large interaction networks. We take a novel approach using protein-chemical interactions derived from 3D structures. The basic premise is that two proteins sharing a common bound chemical will likely share others. We use protein and chemical superimpositions and physical tests of chemical-protein compatibility to identify the most likely candidates among the nearly one million potential interactions. We show for a benchmark that known protein-chemical structures are reconstructed with good accuracy and sometimes via very different proteins and chemicals. We make thousands of confident predictions, including structures for known protein-drug interactions lacking a structure (e.g. topoisomerase-2/radicicol) and many new interactions. The number of confident predictions grows faster than the number of known structures, suggesting that this approach will play a key role in completing the protein-chemical interaction repertoire.