Tomaz Curk

Orange: from experimental machine learning to interactive data mining

Orange (www.ailab.si/orange) is a suite for machine learning and data mining. For researchers in machine learning, Orange offers scripting to easily prototype new algorithms and experimental procedures. For explorative data analysis, it provides a visual programming framework with emphasis on interactions and creative combinations of visual components.

iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

Transcriptome-wide analysis of protein-RNA interactions predicts the dual splicing effects of TIA proteins, showing that their local enhancing function is associated with diverse distal splicing silencing effects.

Microarray data mining with visual programming

UNLABELLED Visual programming offers an intuitive means of combining known analysis and visualization methods into powerful applications. The system presented here enables users who are not programmers to manage microarray and genomic data flow and to customize their analyses by combining common data analysis tools to fit their needs. AVAILABILITY http://www.ailab.si/supp/bi-visprog SUPPLEMENTARY INFORMATION http://www.ailab.si/supp/bi-visprog.

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2. Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12. Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

Conserved developmental transcriptomes in evolutionarily divergent species

BackgroundEvolutionarily divergent organisms often share developmental anatomies despite vast differences between their genome sequences. The social amoebae Dictyostelium discoideum and Dictyostelium purpureum have similar developmental morphologies although their genomes are as divergent as those of man and jawed fish.ResultsHere we show that the anatomical similarities are accompanied by extensive transcriptome conservation. Using RNA sequencing we compared the abundance and developmental regulation of all the transcripts in the two species. In both species, most genes are developmentally regulated and the greatest expression changes occur during the transition from unicellularity to multicellularity. The developmental regulation of transcription is highly conserved between orthologs in the two species. In addition to timing of expression, the level of mRNA production is also conserved between orthologs and is consistent with the intuitive notion that transcript abundance correlates with the amount of protein required. Furthermore, the conservation of transcriptomes extends to cell-type specific expression.ConclusionsThese findings suggest that developmental programs are remarkably conserved at the transcriptome level, considering the great evolutionary distance between the genomes. Moreover, this transcriptional conservation may be responsible for the similar developmental anatomies of Dictyostelium discoideum and Dictyostelium purpureum.

Polymorphic members of the lag gene family mediate kin discrimination in Dictyostelium.

Self and kin discrimination are observed in most kingdoms of life and are mediated by highly polymorphic plasma membrane proteins. Sequence polymorphism, which is essential for effective recognition, is maintained by balancing selection. Dictyostelium discoideum are social amoebas that propagate as unicellular organisms but aggregate upon starvation and form fruiting bodies with viable spores and dead stalk cells. Aggregative development exposes Dictyostelium to the perils of chimerism, including cheating, which raises questions about how the victims survive in nature and how social cooperation persists. Dictyostelids can minimize the cost of chimerism by preferential cooperation with kin, but the mechanisms of kin discrimination are largely unknown. Dictyostelium lag genes encode transmembrane proteins with multiple immunoglobulin (Ig) repeats that participate in cell adhesion and signaling. Here, we describe their role in kin discrimination. We show that lagB1 and lagC1 are highly polymorphic in natural populations and that their sequence dissimilarity correlates well with wild-strain segregation. Deleting lagB1 and lagC1 results in strain segregation in chimeras with wild-type cells, whereas elimination of the nearly invariant homolog lagD1 has no such consequences. These findings reveal an early evolutionary origin of kin discrimination and provide insight into the mechanism of social recognition and immunity.

Comprehensive Identification of RNA-Binding Domains in Human Cells

Summary Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells.

dictyExpress: a Dictyostelium discoideum gene expression database with an explorative data analysis web-based interface

BackgroundBioinformatics often leverages on recent advancements in computer science to support biologists in their scientific discovery process. Such efforts include the development of easy-to-use web interfaces to biomedical databases. Recent advancements in interactive web technologies require us to rethink the standard submit-and-wait paradigm, and craft bioinformatics web applications that share analytical and interactive power with their desktop relatives, while retaining simplicity and availability.ResultsWe have developed dictyExpress, a web application that features a graphical, highly interactive explorative interface to our database that consists of more than 1000 Dictyostelium discoideum gene expression experiments. In dictyExpress, the user can select experiments and genes, perform gene clustering, view gene expression profiles across time, view gene co-expression networks, perform analyses of Gene Ontology term enrichment, and simultaneously display expression profiles for a selected gene in various experiments. Most importantly, these tasks are achieved through web applications whose components are seamlessly interlinked and immediately respond to events triggered by the user, thus providing a powerful explorative data analysis environment.ConclusiondictyExpress is a precursor for a new generation of web-based bioinformatics applications with simple but powerful interactive interfaces that resemble that of the modern desktop. While dictyExpress serves mainly the Dictyostelium research community, it is relatively easy to adapt it to other datasets. We propose that the design ideas behind dictyExpress will influence the development of similar applications for other model organisms.

Developmentally Regulated DNA Methylation in Dictyostelium discoideum

ABSTRACT Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.

Identification of evolutionarily conserved exons as regulated targets for the splicing activator Tra2β in development

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.