Alternative Splicing and Protein Structure Evolution

In den letzten Jahren gab es in verschiedensten Bereichen der Biologie einen dramatischen Anstieg verfügbarer, experimenteller Daten. Diese erlauben zum ersten Mal eine detailierte Analyse der Funktionsweisen von zellulären Komponenten wie Genen und Proteinen, die Analyse ihrer Verknüpfung in zellulären Netzwerken sowie der Geschichte ihrer Evolution. Insbesondere der Bioinformatik kommt hier eine wichtige Rolle in der Datenaufbereitung und ihrer biologischen Interpretation zu. In der vorliegenden Doktorarbeit werden zwei wichtige Bereiche der aktuellen bioinformatischen Forschung untersucht, nämlich die Analyse von Proteinstrukturevolution und Ähnlichkeiten zwischen Proteinstrukturen, sowie die Analyse von alternativem Splicing, einem integralen Prozess in eukaryotischen Zellen, der zur funktionellen Diversität beiträgt. Insbesondere führen wir mit dieser Arbeit die Idee einer kombinierten Analyse der beiden Mechanismen (Strukturevolution und Splicing) ein. Wir zeigen, dass sich durch eine kombinierte Betrachtung neue Einsichten gewinnen lassen, wie Strukturevolution und alternatives Splicing sowie eine Kopplung beider Mechanismen zu funktioneller und struktureller Komplexität in höheren Organismen beitragen. Die in der Arbeit vorgestellten Methoden, Hypothesen und Ergebnisse können dabei einen Beitrag zu unserem Verständnis der Funktionsweise von Strukturevolution und alternativem Splicing bei der Entstehung komplexer Organismen leisten wodurch beide, traditionell getrennte Bereiche der Bioinformatik in Zukunft voneinander profitieren können

Systematic comparison of SCOP and CATH: a new gold standard for protein structure analysis

Background: SCOP and CATH are widely used as gold standards to benchmark novel protein structure comparison methods as well as to train machine learning approaches for protein structure classification and prediction. The two hierarchies result from different protocols which may result in differing classifications of the same protein. Ignoring such differences leads to problems when being used to train or benchmark automatic structure classification methods. Here, we propose a method to compare SCOP and CATH in detail and discuss possible applications of this analysis. Results: We create a new mapping between SCOP and CATH and define a consistent benchmark set which is shown to largely reduce errors made by structure comparison methods such as TM-Align and has useful further applications, e. g. for machine learning methods being trained for protein structure classification. Additionally, we extract additional connections in the topology of the protein fold space from the orthogonal features contained in SCOP and CATH. Conclusion: Via an all-to-all comparison, we find that there are large and unexpected differences between SCOP and CATH w.r.t. their domain definitions as well as their hierarchic partitioning of the fold space on every level of the two classifications. A consistent mapping of SCOP and CATH can be exploited for automated structure comparison and classification. Availability: Benchmark sets and an interactive SCOP-CATH browser are available at http://www.bio.ifi.lmu.de/SCOPCath

AutoPSI: a database for automatic structural classification of protein sequences and structures

In protein research, structural classifications of protein domains provided by databases such as SCOP play an important role. However, as such databases have to be curated and prepared carefully, they update only up to a few times per year, and in between newly entered PDB structures cannot be used in cases where a structural classification is required. The Automated Protein Structure Identification (AutoPSI) database delivers predicted SCOP classifications for several thousand yet unclassified PDB entries as well as millions of UniProt sequences in an automated fashion. In order to obtain predictions, we make use of two recently published methods, namely AutoSCOP (sequence-based) and Vorolign (structure-based) and the consensus of both. With our predictions, we bridge the gap between SCOP versions for proteins with known structures in the PDB and additionally make structure predictions for a very large number of UniProt proteins. AutoPSI is freely accessible at http://www.bio.ifi.lmu.de/AutoPSIDB

Alternative splicing and protein structure evolution

Alternative splicing is thought to be one of the major sources for functional diversity in higher eukaryotes. Interestingly, when mapping splicing events onto protein structures, about half of the events affect structured and even highly conserved regions i.e. are non-trivial on the structure level. This has led to the controversial hypothesis that such splice variants result in nonsense-mediated mRNA decay or non-functional, unstructured proteins, which do not contribute to the functional diversity of an organism. Here we show in a comprehensive study on alternative splicing that proteins appear to be much more tolerant to structural deletions, insertions and replacements than previously thought. We find literature evidence that such non-trivial splicing isoforms exhibit different functional properties compared to their native counterparts and allow for interesting regulatory patterns on the protein network level. We provide examples that splicing events may represent transitions between different folds in the protein sequence–structure space and explain these links by a common genetic mechanism. Taken together, those findings hint to a more prominent role of splicing in protein structure evolution and to a different view of phenotypic plasticity of protein structures

Manananggal - a novel viewer for alternative splicing events

Background: Alternative splicing is an important cellular mechanism that can be analyzed by RNA sequencing. However, identification of splicing events in an automated fashion is error-prone. Thus, further validation is required to select reliable instances of alternative splicing events (ASEs). There are only few tools specifically designed for interactive inspection of ASEs and available visualization approaches can be significantly improved. Results: Here, we present Manananggal, an application specifically designed for the identification of splicing events in next generation sequencing data. Manananggal includes a web application for visual inspection and a command line tool that allows for ASE detection. We compare the sashimi plots available in the IGV Viewer, the DEXSeq splicing plots and SpliceSeq to the Manananggal interface and discuss the advantages and drawbacks of these tools. We show that sashimi plots (such as those used by the IGV Viewer and SpliceSeq) offer a practical solution for simple ASEs, but also indicate short-comings for highly complex genes. Conclusion: Manananggal is an interactive web application that offers functions specifically tailored to the identification of alternative splicing events that other tools are lacking. The ability to select a subset of isoforms allows an easier interpretation of complex alternative splicing events. In contrast to SpliceSeq and the DEXSeq splicing plot, Manananggal does not obscure the gene structure by showing full transcript models that makes it easier to determine which isoforms are expressed and which are not

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation

Vorescore—fold recognition improved by rescoring of protein structure models

Summary: The identification of good protein structure models and their appropriate ranking is a crucial problem in structure prediction and fold recognition. For many alignment methods, rescoring of alignment-induced models using structural information can improve the separation of useful and less useful models as compared with the alignment score. Vorescore, a template-based protein structure model rescoring system is introduced. The method scores the model structure against the template used for the modeling using Vorolign. The method works on models from different alignment methods and incorporates both knowledge from the prediction method and the rescoring

ProSAS: a database for analyzing alternative splicing in the context of protein structures

Alternative splicing is known to be one of the major sources for functional diversity in higher eukaryotes. Several splicing isoforms have been characterized in the literature that play important roles in cellular processes like apoptosis or signal transduction pathways. Splicing events can often be detected on the mRNA level by large-scale cDNA or EST experiments and such data is collected and annotated in several databases. Nevertheless, the effects of splicing on the structure of a protein are largely unknown. The ProSAS (Protein Structure and Alternative Splicing) database fills this gap and provides a unified resource for analyzing effects of alternative splicing events in the context of protein structures. ProSAS comprehensively annotates and models protein structures for several Ensembl genomes as well as SwissProt entries harbouring splicing events. Alternative isoforms annotated in Ensembl or SwissProt can be analyzed on the protein structure and protein function level using an intuitive user interface that provides several features and tools for a structure-based analysis of alternative splicing events. The ProSAS database is freely accessible at http://www.bio.ifi.lmu.de/ProSAS