2013 Rustbelt RNA Meeting
The MDM2 oncogene is a regulator of the tumor suppressor protein p53. Under stress and in cancer, MDM2 is alternatively spliced into numerous isoforms. MDM2-ALT1 is comprised of exons 3 and 12, and we have shown that MDM2-ALT1 is expressed in 85% of rhabdomyosarcomas (RMS) and is also predictive of high-grade metastatic disease. There is therefore a critical need to understand MDM2 alternative splicing and to modulate its splicing in cancer.
In order to study the alternative splicing of MDM2 we have developed a damage-inducible minigene system. The MDM2 3-11-12 minigene recapitulates the splicing of the endogenous gene by excluding exon 11 under genotoxic stress. In order to identify cis-elements present on the pre-mRNA we performed chimeric swaps with elements from a non-damage-responsive minigene and showed that exon 11 of MDM2 is sufficient to confer damage-inducible alternative splicing in a heterologous context. Using ESEfinder 3.0 we identified conserved consensus sequences for splicing regulator SF2/ASF in exon 11 of MDM2. Therefore, we hypothesize that SF2/ASF is responsible for damage-induced alternative splicing of MDM2 under genotoxic stress.
We examined the effects of SF2/ASF on MDM2 alternative splicing in vitro through binding and splicing assays using the wild-type and mutant minigenes. Additionally we performed SF2/ASF knockdown and overexpression studies to elucidate its role in stress-induced splicing changes. We report that SF2/ASF promotes the exclusion of exon 11 under damage. Furthermore, antisense oligonucleotides (ASOs) targeting SF2/ASF binding sites push endogenous MDM2 splicing toward the full-length isoform. Importantly, we also observe elevated SF2/ASF expression in RMS samples concomitant with MDM2-ALT1.
Our results provide valuable insight into the regulation of damage-induced MDM2 alternative splicing by SF2/ASF; modulation of its alternative splicing through ASOs therefore presents a potential target for anticancer therapy.
Keywords: MDM2, Alternative Splicing, SF2ASF