Genomic Views of Splicing Regulation

Understanding how RNA-binding proteins (RBPs) determine the splicing outcome is fundamental to human biology and disease. Splicing is regulated by activators and repressors that recognise cis-elements at their target exons. However, it is clear that RBP recruitment is not solely dependent on the underlying RNA sequence, but is strongly shaped by the interactive environment. Means of interaction include the direct competition or cooperative recruitment of RBPs as well as modulations of secondary structure (Figure 1). However, the general rules that govern the interactive assembly of pre-messenger ribonucleoprotein (pre-mRNP) complexes remain to be established.

Our lab unites a systemic picture of pre-mRNP assembly during splicing with the molecular understanding of the underlying processes. We employ cutting-edge genomic RNA biology techniques, such as iCLIP and RNA-seq, in combination with biochemical and genetic tools. An important aspect of our work is the computational analysis and integration of our genome-wide data sets, and we closely collaborate with bioinformatics experts.

Figure 1. Early spliceosome assembly on a schematic intron. The binding of U2AF65 to the polypyrimidine tract at the 3' splice site (i.e. the 3' end of the intron) acts as an important checkpoint, at which signals of multiple splicing activators and repressors are integrated.


3' Splice-site Definition

A major focus of our lab is the early events of spliceosome assembly at the 3' splice site. An important checkpoint is the binding of the splicing factor U2AF65 to the polypyrimidine tract immediately upstream of the 3' splice site, which we can beautifully monitor on a genome-wide scale with our iCLIP experiments (Figure 2). U2AF65 binding is regulated by several additional splicing factors, which makes it a prime example of how mRNP assembly is shaped by competitive and synergetic interactions.

Figure 2. (A) Overview of the iCLIP protocol (König et al., 2012). Crosslinked protein/RNA complexes are purified from cells. Upon reverse transcription, high-throughput sequencing of the truncated cDNA molecules allows to pinpoint the nucleotide within the RNA that was crosslinked to the RBP. (B) Genome browser view of an example gene (CD55; RefSeq) showing U2AF65 iCLIP data (purple) and RNA-seq data (green). U2AF65 shows strong peaks of binding in front of all exons.


hnRNP Proteins in Splicing Regulation

Of particular interest to our lab are a family of RBPs called heterogeneous nuclear ribonucleoproteins (hnRNPs). Rivalling histones in their abundance, hnRNP proteins have been described to form hnRNP particles, which have - in analogy to nucleosomes - been referred to as "ribonucleosomes". Their high abundance and presence along most transcripts suggests them as major players in guiding the binding and function of other RBPs.

Splicing of Alu Elements and Genetic Disease

The increasing number of reported genetic disorders that are caused by erroneous splicing of Alu elements, illustrates that the enormous amount of Alu elements poses a serious threat to the normal function of human cells. We recently discovered that hnRNP C, the core component of hnRNP particles, acts as a general repressor of Alu elements and blocks their recognition through the splicing machinery. We are now investigating how this block is overcome in genetic disease.

Interested PhD students, postdocs or master students are encouraged to apply.