Background

The Farnesoid X Receptor (FXR) is a nuclear receptor activated by bile acids. Upon activation, FXR regulates transcription of genes involved in bile acid, lipid, glucose and amino acid metabolism and is therefore an appealing therapeutic target in steatotic liver disease. The predominant FXR isoforms in the liver are FXRα1 and FXRa2, which differ in a four amino acid insertion (MYTG) of the DNA binding domain resulting from the use of an alternative 5’-splice site. In steatotic liver disease patients, the isoform distribution is shifted towards FXRa1/2, while fasting and exercise results in more FXRa2/1. Both isoforms bind to IR-1 (inverted repeat 1) DNA motifs, but FXRα2 additionally binds  to  ER-2 (everted repeat 2) DNA motifs, resulting in activation of additional transcriptional programs, including inhibiting lipogenesis. Therefore, FXRα2 is responsible for most metabolic effects of FXR in the liver. We hypothesize that specifically increasing FXRα2 expression results in more ER-2-mediated transcription, thereby improving liver metabolism. Therefore, we aim to unravel the molecular mechanisms that control the alternative splicing of FXR.

Results

We used publicly available eCLIP datasets of RNA binding proteins to identify enriched proteins in the alternative 5’-splice site and found pre-mRNA processing factor 8 (PRPF8) to be highly enriched. Therefore, we generated HepG2 and HEK293T cell lines with lentiviral-mediated PRPF8 knockdown and overexpression. We will use different methods to assess the FXR isoform ratio upon PRPF8 silencing and overexpression. If PRPF8 affects FXR splicing, we will examine ER-2-mediated gene transcription and subsequently liver metabolism.

Conclusion

By gaining more insights in the molecular mechanisms that control FXR alternative splicing we aim to specifically increase FXRα2 expression with the ultimate aim to improve therapeutic efficacy of FXR agonism in steatotic liver disease.