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The last decade has seen the development of a variety of so-called `next-generation' sequencing (NGS) technologies, that have revolutionized the field of genomics and post-genomics. In cancer research area, as NGS permeates the cancer biomarker realm and cost of NGS declines, hospitals and clinics will deploy deep sequencing as a means to personalize cancer diagnostics and therapeutics. Here we provide a new automated high-performance bioinformatics web platform, ODESSA (Online Deep Exome Sequencing Software Analysis), developed for targeting genes at high coverage through deep sequencing with the maximum usability, and focused on rational diagnosis of targeted therapies.

To understand the general features of the transcriptional response of mammalian cells to ionizing radiation we performed a meta-analysis of a transcriptomic dataset referring to 202 different irradiation conditions. Since there is a growing interest in the peculiarities of the response to high-LET versus low-LET irradiation, we enriched the dataset with high-LET conditions, Our results showed that the most general feature of the transcriptional response of mammalian cells to irradiation is the up-regulation CDKN1A which is universally observed in cancer and non-cancer cell lines and in highly proliferating tissues, such as skin, lymph node and blood. This induction does not require a wild-type P53 and is poorly radiation dose dependent. Moreover, it is observed at various times following both high- and low-LET irradiation. Among the genes mostly down-regulated by high-Let irradiation we found Pif1, a highly conserved protein that functions as DNA helicase and plays key roles in the protection of cells from DNA replication stress and DNA damage and in the maintenance of genome stability. � Accademia Nazionale dei Lincei 2014.

The structure and hydration of reconstituted human topoisomerase I comprising the core and the carboxyl-terminal domains in covalent complex with 22-basepair DNA duplex has been investigated by molecular dynamics simulation. The structure and the intermolecular interactions were found to be well maintained over the simulation. The complex displays a high degree of flexibility of the contact area, confirmed by the presence of numerous water-mediated protein-DNA hydrogen bonds comparable in quantity and distribution to the direct ones. The interaction between the enzyme and the solvent also provides the key for interpreting the experimental reduction of activity or affinity observed upon single residue mutation. Finally, four long lasting water molecules are observed in the proximity of the active site, one of which in the appropriate position to accept a proton from the active Tyr723.

There is growing evidence that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) plays a role in breast cancer prevention and survival. It elicits a variety of antitumor activities like controlling cellular differentiation, proliferation and angiogenesis. Most of its biological effects are exerted via its nuclear receptor which acts as a transcriptional regulator. Here, we carried out a genome-wide investigation of the primary transcriptional targets of 1α,25(OH)2D3 in breast epithelial cancer cells using RNA-Seq technology. We identified early transcriptional targets of 1α,25(OH)2D3 involved in adhesion, growth regulation, angiogenesis, actin cytoskeleton regulation, hexose transport, inflammation and immunomodulation, apoptosis, endocytosis and signaling. Furthermore, we found several transcription factors to be regulated by 1α,25(OH)2D3 that subsequently amplify and diversify the transcriptional output driven by 1α,25(OH)2D3 leading finally to a growth arrest of the cells. Moreover, we could show that 1α,25(OH)2D3 elevates the trimethylation of histone H3 lysine 4 at several target gene promoters. Our present transcriptomic analysis of differential expression after 1α,25(OH)2D3 treatment provides a resource of primary 1α,25(OH)2D3 targets that might drive the antiproliferative action in breast cancer epithelial cells.

It is well known that in some places of the human genome one finds a variable number of tandem repeats of trinucleotides; it is now commonly acknowledged that in many cases an excessive expansion of such a number is the cause of nervous system diseases. Moreover there exist cases of genetic disorders linked with loci where a variable number of tandem repeats of sequences longer than three bases has been found. The abnormal number of these repeats in few cases has been associated with the onset of the disease. Considering the above facts, we have performed an extensive study of published sequences of genes connected with various diseases. We have examined, inside or near those genes, all possible tandem repeats. The analysis has led to the detection of a large number of repeats of both triplets and longer sequences, many of which, as far as we know, had not been pointed out before. The results of our analysis lead us to put forward the hypothesis that in more cases than those till now established, a variable number of tandem repeats of generic sequences, not only of triplets, could be associated with disease onset. Finally we suggest to allocate experimental researches for all the possible tandem repeats and their possible correlation with the neurodegenerative disorders and with other kinds of syndromes.

Background The advent of massively parallel sequencing technologies (Next Generation Sequencing, NGS) profoundly modified the landscape of human genetics. In particular, Whole Exome Sequencing (WES) is the NGS branch that focuses on the exonic regions of the eukaryotic genomes; exomes are ideal to help us understanding high-penetrance allelic variation and its relationship to phenotype. A complete WES analysis involves several steps which need to be suitably designed and arranged into an efficient pipeline. Managing a NGS analysis pipeline and its huge amount of produced data requires non trivial IT skills and computational power. Results Our web resource WEP (Whole-Exome sequencing Pipeline web tool) performs a complete WES pipeline and provides easy access through interface to intermediate and final results. The WEP pipeline is composed of several steps: 1) verification of input integrity and quality checks, read trimming and filtering; 2) gapped alignment; 3) BAM conversion, sorting and indexing; 4) duplicates removal; 5) alignment optimization around insertion/deletion (indel) positions; 6) recalibration of quality scores; 7) single nucleotide and deletion/insertion polymorphism (SNP and DIP) variant calling; 8) variant annotation; 9) result storage into custom databases to allow cross-linking and intersections, statistics and much more. In order to overcome the challenge of managing large amount of data and maximize the biological information extracted from them, our tool restricts the number of final results filtering data by customizable thresholds, facilitating the identification of functionally significant variants. Default threshold values are also provided at the analysis computation completion, tuned with the most common literature work published in recent years. Conclusions Through our tool a user can perform the whole analysis without knowing the underlying hardware and software architecture, dealing with both paired and single end data. The interface provides an easy and intuitive access for data submission and a user-friendly web interface for annotated variant visualization. Non-IT mastered users can access through WEP to the most updated and tested WES algorithms, tuned to maximize the quality of called variants while minimizing artifacts and false positives. The web tool is available at the following web address: http://www.caspur.it/wep

Our goal was to identify genes showing a general transcriptional response to irradiation in mammalian cells and to analyze their response in function of dose, time and quality of irradiation and of cell type.We used a modified MIAME (Minimal Information About Microarray Experiments) protocol to import microarray data from 177 different irradiation conditions in the Radiation Genes database and performed cut-off-based selections and hierarchical gene clustering.We identified a set of 29 genes which respond to a wide range of irradiation conditions in different cell types and tissues. Functional analysis of the negatively modulated genes revealed a dominant signature of mitotic cell cycle regulation which appears both dose and time-dependent. This signature is prominent in cancer cells and highly proliferating tissues but it is strongly attenuated in non cancer cells.The transcriptional response of mammalian cancer cells to irradiation is dominated by a mitotic cell cycle signature both dose and time-dependent. This core response, which is present in cancer cells and highly proliferating tissues such as skin, blood and lymph node, is weaker or absent in non-cancer cells and in liver and spleen. CDKN1A (cyclin-dependent kinase inhibitor 1A) appears as the most generally induced mammalian gene and its response (mostly dose- and time-independent) seems to go beyond the typical DNA damage response.

Myofibrillar myopathies (MFM) represents a group of neuromuscular disorders either familial or sporadic with clinical and genetic heterogeneity. To date, mutations in six genes are known to cause MFM, but a large number of these diseases are caused by so far unidentified gene defects. Within the NMD-chip project, we had previously studied by CGH arrays 21 sporadic patients with a clinical and immunohistochemical diagnosis of myofibrillar myopathy negative at conventional sequencing for small mutations in desmin, α B-crystallin, myotilin, Z-band alternatively spliced PDZ motif containing protein (ZASP) Bcl2-associated athanogene 3 (BAG3). The CGH analysis did not reveal any rearrangement in myofibrillar myopathy known genes. We have therefore run whole exome sequencing (WES) by Illumina GAIIe platform in seven out our 21 negative patients. This approach has allowed us to identified a mean of 60,000 SNPs or DIPs passing standard quality filters. We applied a further bioinformatics filter consisting in 880 candidate genes selected using the MedScan interactome pathway developed by Ariadne Genomics. We looked at SNPs or DIPs occurring in heterozygosity (according with a dominant model of inheritance) within all these genes. All SNPs already reported in the dbSNP database were excluded. This analysis led to the identification of a mean of 10 variations/patient. We detected (in heterozygosis) a nonsense and a missense variations in Filamin C, and 3 missense variations in three unrelated patients, within the Titin gene. Mutations in this huge gene, are known to cause a recessive form of LGMD (LGMD2J), but have not been previously related to MFMs. We have also identified stop mutations in other three novel possibly causative genes. All variations are in course of validation. We demonstrate here that WES, following facilitation of both molecular and bioinformatics procedures, may represent a "second step" powerful diagnostic tool in NMDs with high genetic heterogeneity.

The MITOchondrial genome database of metaZOAns (MitoZoa) is a public resource for comparative analyses of metazoan mitochondrial genomes (mtDNA) at both the sequence and genomic organizational levels. The main characteristics of the MitoZoa database are the careful revision of mtDNA entry annotations and the possibility of retrieving gene order and non-coding region (NCR) data in appropriate formats. The MitoZoa retrieval system enables basic and complex queries at various taxonomic levels using different search menus. MitoZoa 2.0 has been enhanced in several aspects, including: a re-annotation pipeline to check the correctness of protein-coding gene predictions; a standardized annotation of introns and of precursor ORFs whose functionality is post-transcriptionally recovered by RNA editing or programmed translational frameshifting; updates of taxon-related fields and a BLAST sequence similarity search tool. Database novelties and the definition of standard mtDNA annotation rules, together with the user-friendly retrieval system and the BLAST service, make MitoZoa a valuable resource for comparative and evolutionary analyses as well as a reference database to assist in the annotation of novel mtDNA sequences. MitoZoa is freely accessible at http://www.caspur.it/mitozoa. © The Author(s) 2011. Published by Oxford University Press.

De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.

Summary: ExpEdit is a web application for assessing RNA editing in human at known or user-specified sites supported by transcript data obtained by RNA-Seq experiments. Mapping data (in SAM/BAM format) or directly sequence reads [in FASTQ/short read archive (SRA) format] can be provided as input to carry out a comparative analysis against a large collection of known editing sites collected in DARNED database as well as other user-provided potentially edited positions. Results are shown as dynamic tables containing University of California, Santa Cruz (UCSC) links for a quick examination of the genomic context. Availability: ExpEdit is freely available on the web at http://www.caspur.it/ExpEdit/. Contact: graziano.pesole@biologia.uniba.it Supplementary information: Supplementary data are available at Bioinformatics online.

The transcription factor interferon regulatory factor 6 (IRF6) regulates craniofacial development and epidermal proliferation. We recently showed that IRF6 is a component of a regulatory feedback loop that controls the proliferative potential of epidermal cells. IRF6 is transcriptionally activated by p63 and induces its proteasome-mediated down-regulation, thereby limiting keratinocyte proliferative potential. We hypothesized that IRF6 may also be involved in skin carcinogenesis. Hence, we analyzed IRF6 expression in a large series of squamous cell carcinomas (SCCs) and found a strong down-regulation of IRF6 that correlated with tumor invasive and differentiation status. IRF6 down-regulation in SCC cell lines and primary tumors correlates with methylation on a CpG dinucleotide island located in its promoter region. To identify the molecular mechanisms regulating IRF6 potential tumor suppressive activity, we performed a genome-wide analysis by combining ChIP sequencing for IRF6 binding sites and gene expression profiling in primary human keratinocytes after siRNA-mediated IRF6 depletion. We observed dysregulation of cell cycle-related genes and genes involved in differentiation, cell adhesion, and cell–cell contact. Many of these genes were direct IRF6 targets. We also performed in vitro invasion assays showing that IRF6 down-regulation promotes invasive behavior and that reintroduction of IRF6 into SCC cells strongly inhibits cell growth. These results indicate a function for IRF6 in suppression of tumorigenesis in stratified epithelia.

MitoZoa is a relational database collecting curated metazoan entries of complete or nearly complete mitochondrial genomes (mtDNA), specifically designed to assist comparative studies of mitochondrial genome-level features in a given taxon or in congeneric species of Metazoa. The principal novelties of MitoZoa are extensive corrections/improvements of the mtDNA annotations and the possibility of easily searching for data on: (1) gene order, a genomic feature useful as phylogenetic marker; (2) sequence, size and location of non-coding regions, likely containing the regulatory signals for mtDNA replication and transcription; (3) mt features/sequences of congeneric species, where saturation phenomena in nucleotide substitutions and gene order changes are expected to be absent or at least minimal. In addition, MitoZoa allows the exploration of basic mt features such as molecule topology, genetic code, gene content, and compositional parameters of the entire genome. Finally, in order to facilitate downstream analyses of retrieved data, MitoZoa entry lists can be visualized and downloaded in a tabular format, while sequences and gene order data are provided in FASTA and FASTA-like formats, respectively. The MitoZoa database is available at http://www.caspur.it/mitozoa .

The analysis of the great extent of data generated by using DNA microarrays technologies has shown that the transcriptional response to radiation can be considerably different depending on the quality, the dose range and dose rate of radiation, as well as the timing selected for the analysis. At present, it is very difficult to integrate data obtained under several experimental conditions in different biological systems to reach overall conclusions or build regulatory models which may be tested and validated. In fact, most available data is buried in different websites, public or private, in general or local repositories or in files included in published papers; it is often in various formats, which makes a wide comparison even more difficult. The Radiation Genes Database (http://www.caspur.it/RadiationGenes) collects microarrays data from various local and public repositories or from published papers and supplementary materials. The database classifies it in terms of significant variables, such as radiation quality, dose, dose rate and sampling timing, as to provide user-friendly tools to facilitate data integration and comparison.

Motivation: Alternative splicing has recently emerged as a key mechanism responsible for the expansion of transcriptome and proteome complexity in human and other organisms. Although several online resources devoted to alternative splicing analysis are available they may suffer from limitations related both to the computational methodologies adopted and to the extent of the annotations they provide that prevent the full exploitation of the available data. Furthermore, current resources provide limited query and download facilities. Results: ASPicDB is a database designed to provide access to reliable annotations of the alternative splicing pattern of human genes and to the functional annotation of predicted splicing isoforms. Splice-site detection and full-length transcript modeling have been carried out by a genome-wide application of the ASPic algorithm, based on the multiple alignments of gene-related transcripts (typically a Unigene cluster) to the genomic sequence, a strategy that greatly improves prediction accuracy compared to methods based on independent and progressive alignments. Enhanced query and download facilities for annotations and sequences allow users to select and extract specific sets of data related to genes, transcripts and introns fulfilling a combination of user-defined criteria. Several tabular and graphical views of the results are presented, providing a comprehensive assessment of the functional implication of alternative splicing in the gene set under investigation. ASPicDB, which is regularly updated on a monthly basis, also includes information on tissue-specific splicing patterns of normal and cancer cells, based on available EST sequences and their library source annotation. Availability: www.caspur.it/ASPicDB Contact: graziano.pesole@biologia.uniba.it Supplementary information: Supplementary data are available at Bioinformatics online.

Identification and molecular typing of environmental isolates by molecular techniques requires knowledge of the genetic characteristics of the microbe species being examined. The introduction of automated sequences has greatly speeded up the entire sequencing process as well as improved the accuracy of the collected information. Bioinformatics tools have become indispensable not only for setting up research studies, but also for storing, organizing and managing enormous quantities of sequencing data. Despite its great advantages, the use of bioinformatics is hindered by difficulties in learning how to use its software tools. The GenEnv database was developed to provide operators involved in biological risk management with a user-friendly tool for sequence analysis. Presently, there are over 20.000 sequence records, and over 9000 bacterial species represented in the database. The initial gene set comprises rDNA16S, rpoB, gyrB. The system allows sequence-driven microbe identification as well as the development of study protocols for research on specific microbe species. Nucleotide sequences are represented graphically. The GenEnv database was designed as a tool for public health operators but also offers wide prospects for scientific research.

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource - available at http://www.caspur.it/ASPIC/ - is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. © The Author 2006. Published by Oxford University Press. All rights reserved.

Summary: GenoMiner is a software tool that searches for regions of similarity between user-submitted genome or transcript sequences and user-specified whole genome assemblies. The program then identifies conserved sequence tags (CSTs) in these homologous regions and provides a prediction of their coding or non-coding nature. The analysis is carried out through three steps: (1) definition of sequence regions homologous to the query sequence in the selected target genomes by a fast BLAT alignment; (2) identification of CSTs by a more sensitive BLAST-like alignment between the query and the homologous regions in the target genomes and (3) assessment of the coding or non-coding nature of detected CSTs through the computation of a suitable coding potential score. GenoMiner allows the user to search the query sequence against a number of vertebrate genome assemblies in a single run providing a user-friendly graphical output. Availability: . GenoMiner software and documentation is available from the authors upon request. Contact: graziano.pesole@unimi.it

The identification and characterization of genome tracts that are highly conserved across species during evolution may contribute significantly to the functional annotation of whole-genome sequences. Indeed, such sequences are likely to correspond to known or unknown coding exons or regulatory motifs. Here, we present a web server implementing a previously developed algorithm that, by comparing user-submitted genome sequences, is able to identify statistically significant conserved blocks and assess their coding or noncoding nature through the measure of a coding potential score. The web tool, available at http://www.caspur.it/CSTminer/, is dynamically interconnected with the Ensembl genome resources and produces a graphical output showing a map of detected conserved sequences and annotated gene features.

Several genes encoding salivary components of the mosquito Anopheles gambiae were identified using a selective trapping approach. Among these, five corresponded to genes expressed specifically in female glands and their role may possibly be linked to blood-feeding. Our collection included a fourth member of the D7 protein family and two polypeptides that showed weak similarity to anti-coagulants from distantly related species. Moreover, we identified two additional members of a novel group of proteins that we named glandins. The isolation of tissue-specific genes represents a first step toward a deeper molecular analysis of mosquito salivary secretions.

We consider the Hill coefficient obtained from concentration–response curves and we stress the differences between the use of binding and the use of current for the response. We first show that in order to estimate the cooperativity of binding sites one must use the coefficient obtained from the binding curves and not that obtained from the current curves. For what concerns the estimation of the minimum number of bound ligands needed to activate the channel, while the coefficient obtained from the binding is simply not applicable, we show that the coefficient obtained from the current is not appropriate. We finally introduce a novel parameter that predicts the number of ligand molecules required to open the channel from current measurements at very low ligand concentration. The above considerations are exemplified by a few theoretical models.

We studied a family of four with a form of juvenile Parkinson, cognitive impairment, ataxia, and obesity, with variable clinical severity. Many genes associated to these diseases were excluded with conventional sequencing: SCA1, SCA2, SCA3, SCA6 and DRPLA for the spinocerebellar ataxias, GM1 and GM2 for the adrenoleukodystrophy, GCH1 for dopa-responsive dystonia and the linkage analysis excluded the Parkinson genes PARK2, PARK3 and PARK7. For both affected patients we hypothesized a neurological disease with multisystem involvement that was genetically determined. We performed the whole exome sequencing analysis by Illumina GAIIe platform on all family members except the affected sister. We identified a mean of 30,000 SNPs or DIPs passing a quality filters (coverage >10, ambigously mapped reads per variant 50, variant confidence/consensus quality >1.5). We elaborated a list of 143 genes that are frankly or putatively associated to Parkinson, obesity or spinocerebellar ataxia and adopted this list as a filter applied to the WES analysis, considering SNPs only in homozygosity or in compound heterozygosity (dbSNPs not excluded). This analysis allowed us to detect 82 variations within 41 genes in homozygosity and 134 variations in compound heterozygosity within 39 genes. We filtered these identified variations in family’s members or in other samples present in the same run; this investigation greatly reduced the numbers of variations, being now 12 homozygous and 54 the compound heterozygous. The variations identified are in course of technical validation as possible mutations in novel genes causing the peculiar phenotype in this family.

We identified a Duchenne-like female clinically evaluated as a severely affected symptomatic DMD carrier. Carrier status was confirmed by evidence of mosaic pattern of dystrophin expression on muscle biopsy; nevertheless, extensive analysis by MLPA, sequencing, CGH-array and RNA profiling failed to identify any mutation within DMD gene. We performed whole exome sequencing by means of an Illumina GAIIe sequencer in order to identify genetic modifiers that could contribute to the symptomatic phenotype in this female. A total of 23,776 SNPs passed filtering for quality parameters. We further selected variations present in a list of 883 candidate genes belonging to the dystrophin expression regulation network and, applying a dominant model of inheritance for the modifying genes, we considered only heterozygous variations including SNPs already present in the dbSNP database. This allowed us to retain 902 SNPs. We performed RNAseq analysis on her muscle biopsy to restrict the panel of SNPs and integrated exome and transcriptome results to provide critical/confirmatory information about the SNPs identified. Eight out of the 20 SNPs selected in UTR regions were validated by RNAseq data. For non-synonymous variations, 205 out of the 219 SNPs were mapped in genes expressed in muscle and 61 were validated by RNAseq. In total we validated 69 exploratory SNPs. These SNPs are located within genes involved in the sarcolemma and extracellular matrix, cell signalling and, interestingly, chromatin modification. The 69 SNPs are in course of validation in two groups of females: symptomatic (17) and non-symptomatic (18) DMD carriers. Results will be statistically analysed using both multidimensional scaling and discriminate analysis in order to identify those variations able to discriminate between the two phenotypic groups.

Motivations. The pre-mRNA sequences of eukaryotes harbour multiple information layers in addition to the one specifying the aminoacid sequence. Indeed, specific signals regulating the splicing process, RNA folding, capping, polyadenylation, stability and nuclear export, overlap each other and with the coding information. Mutagenesis experiments have highlighted that splicing regulatory elements are scattered in the entire pre-mRNA sequence and that all nucleotide positions are potentially involved in the generation of the specific splicing pattern through the specific interaction with trans-acting RNA-binding proteins. In particular, the identification of cis-regulatory motifs in exonic regions, such as exonic splicing enhancers (ESEs) or silencers (ESSs), superimposed on the coding sequence of the gene can be driven by the observation of purifying selection occurring at synonymous codons [5]. The coordinated binding of combinations of regulatory proteins to their binding sites modulate the expression of specific transcript isoforms in a cell/tissue type-, development stage-, disease- and/or other condition-specific manners and may also promote or repress the formation of the spliceosome, the large (~60S) RNA-protein machinery that catalyzes intron removal. A growing list of mammalian protein factors involved in splicing regulation and their target sites in the pre-mRNA has been reported in the literature [2]. In order to establish a curated and retrievable repository of splicing regulatory factors and target sites for human genes we have recently created SpliceAid [4] that can also be used to find putative regulatory motifs in user submitted sequences. As a further evolution of SpliceAid, we present here SpliceAid-F, a database compilation of splicing regulatory factors and their experimentally validated target RNAs extracted from an exhaustive hand-curated literature search. Methods. For each known splicing factor, cross-links to gene (NCBI Entrez) and protein (Uniprot) IDs, as well as information on the structure of the RNA binding domain, the protein-defect associated disease (MIM), and the interacting proteins (from STRING and IntAct) have been collected. Moreover, we have extracted from the literature the relevant information on the genome coordinates of RNA binding sites (or experimentally validated non-binding sites), type of binding assay, gene information and the context-specific splicing effects of splicing factor binding in terms of exonization or intronization. Furthermore, binding site information have been also reported in the transcript view of ASPicDB [1,3]. Results. SpliceAid-F, currently collects 68 records for splicing regulatory factors and 2489 records for their related binding sites and can be retrieved through a web interface. SpliceAid-F collects in a unique resource heterogeneous information about splicing regulatory proteins, related RNA binding sites and context-specific activity of their interaction. Our database may be a useful resource to retrieve and visualize experimentally known splicing factor binding sites in a gene and to investigate their context where additional binding sites may establish a potential competition or co-regulation. All these information may help to explain the observed splicing pattern as well as the effect of mutations, which if located in functional regulatory elements may generate an aberrant and possibly pathological splicing pattern. This resource can also be useful to develop a new generation of prediction software taking into account all the splicing regulatory element and so allowing to attain a more accurate prediction of splicing patterns. Availability http://www.caspur.it/SpliceAidF References 1. Castrignano T, D'Antonio M, Anselmo A, Carrabino D, D'Onorio De Meo A et al. (2008) ASPicDB: a database resource for alternative splicing analysis. Bioinformatics 24(10):1300-1304. 2. Gabut M, Chaudhry S, Blencowe BJ (2008) SnapShot: The splicing regulatory machinery. Cell 133(1): 192e191. 3. Martelli PL, D'Antonio M, Bonizzoni P, Castrignano T, D'Erchia AM, D'Onorio De Meo P et al. (2011) ASPicDB: a database of annotated transcript and protein variants generated by alternative splicing. Nucleic Acids Res. 39(Database issue):D80-85. 4. Piva F, Giulietti M, Nocchi L, Principato G (2009) SpliceAid: a database of experimental RNA target motifs bound by splicing proteins in humans. Bioinformatics 25(9):1211-1213. 5. Schattner P, Diekhans M (2006) Regions of extreme synonymous codon selection in mammalian genes. Nucleic Acids Res 34(6):1700-1710

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from DrosophilaStaufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD–dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD–dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1–10-ps time scale. This work provides new insight into dsRBD–dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein–nucleic acid interactions.

Background The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). Moreover, the huge volume of data generated by NGS platforms introduces unprecedented computational and technological challenges to efficiently analyze and store sequence data and results. Methods In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Results Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs.