Andrzej Zielezinski

mirEX: a platform for comparative exploration of plant pri-miRNA expression data

mirEX is a comprehensive platform for comparative analysis of primary microRNA expression data. RT–qPCR-based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface. A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Currently, mirEX integrates information about the expression profile of 190 Arabidopsis thaliana pri-miRNAs in seven different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a one-stop solution for Arabidopsis microRNA information. A web-based mirEX interface can be accessed at http://bioinfo.amu.edu.pl/mirex.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs

BackgroundMicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism.DescriptionThe mirEX 2.0 web portal (http://www.combio.pl/mirex) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page.ConclusionsThe mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

Alignment-free sequence comparison: benefits, applications, and tools

Alignment-free sequence analyses have been applied to problems ranging from whole-genome phylogeny to the classification of protein families, identification of horizontally transferred genes, and detection of recombined sequences. The strength of these methods makes them particularly useful for next-generation sequencing data processing and analysis. However, many researchers are unclear about how these methods work, how they compare to alignment-based methods, and what their potential is for use for their research. We address these questions and provide a guide to the currently available alignment-free sequence analysis tools.

5SRNAdb: an information resource for 5S ribosomal RNAs

Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA–protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces.

Agos--a universal web tool for GW Argonaute-binding domain prediction.

MOTIVATION AGO(Argonaute)-binding domains, composed of repeated motifs, in which only binary combinations of tryptophan and glycine are conserved, bind AGO proteins and are essential during RNAi-mediated gene silencing. The amino acid sequence of this domain is extremely divergent and therefore very difficult to detect. Commonly used bioinformatic tools fail to identify tryptophan-glycine and/or glycine-tryptophan motifs (WG/GW) domains and currently there is no publicly available software which can detect these weakly conserved, but functional AGO-binding segments. RESULTS Recently, we have developed an algorithm based on compositional analysis of the amino acid content of the domain. We have demonstrated that the algorithm can be successfully applied for the identification of the new WG/GW proteins in the Arabidopsis genome. Here we introduce Agos (Argonaute-binding domain screener), a novel universal web service for de novo identification of WG/GW domains in protein sequences. The web implementation of the algorithm contains several new features and enhancements: (i) one universal scoring matrix which allows identification of AGO-binding proteins in sequences representing all organisms; (ii) reduction of false positive predictions by improved selectivity of the algorithm; (iii) graphical interface to easily browse the prediction results; and (iv) the option to submit a DNA sequence which will be automatically translated in six frames before running the prediction algorithm. AVAILABILITY Freely available at: http://bioinfo.amu.edu.pl/agos/.

Early origin and adaptive evolution of the GW182 protein family, the key component of RNA silencing in animals.

The GW182 proteins are a key component of the miRNA-dependent post-transcriptional silencing pathway in animals. They function as scaffold proteins to mediate the interaction of Argonaute (AGO)-containing complexes with cytoplasmic poly(A)-binding proteins (PABP) and PAN2-PAN3 and CCR4-NOT deadenylases. The AGO-GW182 complexes mediate silencing of the target mRNA through induction of translational repression and/or mRNA degradation. Although the GW182 proteins are a subject of extensive experimental research in the recent years, very little is known about their origin and evolution. Here, based on complex functional annotation and phylogenetic analyses, we reveal 448 members of the GW182 protein family from the earliest animals to humans. Our results indicate that a single-copy GW182/TNRC6C progenitor gene arose with the emergence of multicellularity and it multiplied in the last common ancestor of vertebrates in 2 rounds of whole genome duplication (WGD) resulting in 3 genes. Before the divergence of vertebrates, both the AGO- and CCR4-NOT-binding regions of GW182s showed significant acceleration in the accumulation of amino acid changes, suggesting functional adaptation toward higher specificity to the molecules of the silencing complex. We conclude that the silencing ability of the GW182 proteins improves with higher position in the taxonomic classification and increasing complexity of the organism. The first reconstruction of the molecular journey of GW182 proteins from the ancestral metazoan protein to the current mammalian configuration provides new insight into development of the miRNA-dependent post-transcriptional silencing pathway in animals.

tRex: A Web Portal for Exploration of tRNA-Derived Fragments in Arabidopsis thaliana

tRNA-derived fragments (tRFs) constitute a new class of short regulatory RNAs that are a product of nascent or mature tRNA processing. tRF sequences have been identified in all domains of life; however, most published research pertains to human, yeast and some bacterial organisms. Despite growing interest in plant tRFs and accumulating evidence of their function in plant development and stress responses, no public, web-based repository dedicated to these molecules is currently available. Here, we introduce tRex (http://combio.pl/trex)-the first comprehensive data-driven online resource specifically dedicated to tRFs in the model plant Arabidopsis thaliana. The portal is based on verified Arabidopsis tRNA annotation and includes in-house-generated and publicly available small RNA sequencing experiments from various tissues, ecotypes, genotypes and stress conditions. The provided web-based tools are designed in a user-friendly manner and allow for seamless exploration of the data that are presented in the form of dynamic tables and cumulative coverage profiles. The tRex database is connected to external genomic and citation resources, which makes it a one-stop solution for Arabidopsis tRF-related research.

ORCAN - a web-based meta-server for real-time detection and functional annotation of orthologs.

Summary: ORCAN (ORtholog sCANner) is a web-based meta-server for one-click evolutionary and functional annotation of protein sequences. The server combines information from the most popular orthology-prediction resources, including 4 tools and 4 online databases. Functional annotation utilizes 5 additional comparisons between the query and identified homologs, including: sequence similarity, protein domain architectures, functional motifs, Gene Ontology term assignments, and a list of associated articles. Furthermore, the server uses a plurality-based rating system to evaluate the orthology relationships and to rank the reference proteins by their evolutionary and functional relevance to the query. Using a dataset of ~1 million true yeast orthologs as a sample reference set, we show that combining multiple orthology-prediction tools in ORCAN increases the sensitivity and precision by 1-2 percent points. Availability: The service is available for free at http://www.combio.pl/orcan/ Contact: wmk@amu.edu.pl Supplementary information: Supplementary data available at Bioinformatics online.Summary: ORCAN (ORtholog sCANner) is a web‐based meta‐server for one‐click evolutionary and functional annotation of protein sequences. The server combines information from the most popular orthology‐prediction resources, including four tools and four online databases. Functional annotation utilizes five additional comparisons between the query and identified homologs, including: sequence similarity, protein domain architectures, functional motifs, Gene Ontology term assignments and a list of associated articles. Furthermore, the server uses a plurality‐based rating system to evaluate the orthology relationships and to rank the reference proteins by their evolutionary and functional relevance to the query. Using a dataset of ˜1 million true yeast orthologs as a sample reference set, we show that combining multiple orthology‐prediction tools in ORCAN increases the sensitivity and precision by 1‐2 percent points. Availability and Implementation: The service is available for free at http://www.combio.pl/orcan/. Contact: wmk@amu.edu.pl Supplementary information: Supplementary data are available at Bioinformatics online.

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells

Abstract Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)–MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer’s disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

Annotation and profiling of barley GLYCOGEN SYNTHASE3/Shaggy-like genes indicated shift in organ-preferential expression

GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 Arabidopsis thaliana GSK genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active GSK genes in barley. We re-evaluated annotation of the GSK genes in the current barley genome (Hv_IBSC_PGSB_v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another GSK, predicted in Hv_IBSC_PGSB_v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in Brachypodium distachyon. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified HvGSK genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of GSKs from the indicated groups showed a shift in organ-preferential expression in A. thaliana and barley illustrating diversification of biological roles of individual HvGSKs in different plant species.