Talk abstracts

Talk on Friday 03:15-03:30pm submitted by Daniel Comiskey

SF2/ASF Modulates Alternative Splicing of MDM2 in Cancer

Daniel F. Comiskey Jr. (Department of Pediatrics, The Ohio State University), Aishwarya G. Jacob (Department of Pediatrics, The Ohio State University), Ravi K. Singh (Department of Pediatrics, The Ohio State University), Aixa S. Tapia-Santos (Department of Pediatrics, The Ohio State University), Dawn S. Chandler (Department of Pediatrics, The Ohio State University)

Abstract:
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