In the post genome era, it has become evident that in higher eukaryotes mechanisms other than regulated transcription play a fundamental role in controlling gene expression. Alternative splicing multiplies the number of possible proteins generated from a single pre-mRNA and therefore is one of the most abundant mechanisms to control protein expression and function posttranscriptionally. Indeed, above 90% of human multi-exon pre-mRNAs are alternatively spliced, many of them in a tissue-, differentiation- or activation-status dependent manner. In addition, splicing patterns are controlled in a species-specific manner and the abundance of alternative splicing correlates with organism complexity, suggesting a role of alternative splicing in the formation of species-specific traits. The ever-growing number of human diseases associated with splicing defects confirms the fundamental impact of (alternative) splicing on generating a functional cell in vivo and demonstrates the severe consequences of its failure.
Even though the existence of cell type and species-specific splicing patterns is well acknowledged, two fundamental questions are only beginning to be addressed: how are particular splicing patterns established and what is their contribution to the functionality and the identity of a cell or a whole organism? We are addressing such questions by investigating the regulation and functional impact of alternative splicing, also coupled to nonsense-mediated decay, in a (body) temperature-dependent manner, during activation of immune cells or in differentiation processes. Two additional research directions we are currently exploring are briefly described below.
The body temperature of homeothermic organisms is tightly controlled in a narrow range. However, we have shown that subtle fluctuations in the core body temperature e.g. due to circadian temperature oscillations, hypothermia or fever, result in global changes in alternative splicing and gene expression.
To address the functionality of body temperature controlled alternative splicing and gene expression we have used extensive analyses of RNA-Seq datasets. Interestingly, we find cancer-associated genes to be differentially expressed and spliced at different temperatures, which may result in a tumor-suppressive environment at higher temperature. We are now addressing the mechanistic basis and functional consequences of temperature-controlled expression of oncogenes and tumor suppressors, which may contribute to a better molecular understanding of thermotherapy as a therapeutic approach.
RNA-Seq and downstream bioinformatics allow the transcriptome-wide quantification of splice site choices. By integrating multiple RNA-Seq datasets from siRNA-mediated perturbations of core components of the spliceosome, we have defined groups of spliceosomal proteins implicated in specific changes in alternative splice site choice. A multi-level bioinformatics analysis pipeline revealed common patterns or features within changed splicing events and led to our current focus on NAGNAG alternative splicing. We now use classical biochemical experiments to gain mechanistic insights into protein-specific changes in splice site selection. After careful validation of bioinformatics predictions using splicing-sensitive RT-PCRs we use bioinformatics-based mutations of cis-regulatory RNA sequences or structure-guided mutations of trans-acting factors for a detailed mechanistic analysis of alternative splice site selection. Ultimately, the combined bioinformatics and biochemical approaches will reveal new paradigms of splice site selection and contribute to understand the incredibly complex regulation of (alternative) splicing.
A, Heatmap confirming specific splicing factor knockdowns within RNA sequencing data.
B, Volcano blot revealing a directed effect of a splicing factor knockdown on alternative acceptor splice site choice (significant events are highlighted by colors).
C, Correlation matrix reveals co-regulation of a certain intron subtype by a subset of splicing factors.