Reserach - Peter 't Hart

Splicing regulation by splicing factors

The discovery of the discontinuous nature of eukaryotic genes, characterized by coding exons interrupted by non-coding introns, marked a pivotal advancement in genetics. Following gene transcription, these non-coding introns are precisely excised, and the coding exons are ligated together in a process known as splicing.

The evolutionary rationale for the existence of this genomic architecture is to allow the organism to alternate the use of exons and yield different messenger RNA isoforms from a single gene known as alternative splicing. Consequently, the resulting proteins possess distinct amino acid sequences and, as a result, can exhibit varied functions. The functional divergence can be profoundly significant, as exemplified by the Bcl-x gene, where a single gene can produce both pro-oncogenic and anti-oncogenic protein isoforms through alternative splicing.

The selection of isoforms is regulated by so-called splicing factors, which can either promote or inhibit the activity of the spliceosome (the machinery that catalyzes the splicing reaction). These factors directly bind pre-mRNA and can affect hundreds or even thousands of splicing events. Mutations or changes in expression levels of these splicing factors can therefore cause a large shift in the proteome and are often observed in oncogenesis.

We use biochemical and biophysical techniques to investigate the interactions between splicing factors and their target RNAs. By delineating the critical elements of these interactions, we lay the groundwork for understanding their formation and, crucially, identifying potential therapeutic targets. However, A significant hurdle in targeting splicing factors is their lack of well-defined binding pockets. To overcome this issue, we use non-small-molecule modalities such as peptides to target these proteins effectively. Our objective is to deploy these inhibitors in cancer cell lines and, using high-throughput sequencing techniques, comprehensively analyze the resulting transcriptome-wide shifts in splicing patterns.

In contrast to inhibition, we also investigate whether we can induce proximity between a splicing factor and non-target RNAs. By doing so, we hope to recover lost binding affinity that occurs after mutation of either the splicing factor or the RNA. These protein-RNA interaction glues (PRIglues) are then expected to correct disrupted alternative splicing patterns.