Poster abstracts

Poster number 8 submitted by Daniel Comiskey

SC35 and SF2/ASF Regulate Stress-Responsive Alternative Splicing of MDM2

Daniel F. Comiskey, Jr. (The Ohio State University), Ravi K. Singh (The Ohio State University), Aixa S. Tapia-Santos (The Ohio State University), Dawn S. Chandler (The Ohio State University)

Abstract:
The MDM2 oncogene encodes a protein that negatively regulates p53 by targeting it for proteasome-mediated degradation. Through the induction of DNA damage and in cancer, MDM2 is alternatively spliced into a variety of isoforms. The MDM2-ALT1 isoform, comprised of exons 3 and 12, is observed in over 85% of rhabdomyosarcomas and is generated in cells in response to genotoxic stress. MDM2-ALT1 lacks a p53 binding domain and abrogates full-length MDM2 from binding p53 by sequestering it. This leads to the stabilization of p53, causing cell cycle arrest and/or apoptosis. Paradoxically, the mouse homolog Mdm2-Alt1 has been shown to accelerate tumorigenesis in a mouse model, indicating a potential role in cancer.

In order to understand the mechanisms by which MDM2 is alternatively spliced, we have developed an in vitro splicing system using MDM2 minigenes and normal and cisplatinum-treated HeLa nuclear extracts. The MDM2 3-11-12 minigene, comprising regions conserved between mouse and human MDM2, predominantly excludes exon 11 under cisplatinum treatment both in vivo and in vitro. Using ESEfinder 3.0, we identified putative binding sites for splicing regulators SC35 and SF2/ASF in exon 11 of the MDM2 minigene. We then performed a series of mutations in the predicted binding sites for SC35 and SF2/ASF. Disrupting the SC35 binding sites in exon 11 leads to greater exclusion of exon 11 under normal conditions. This is consistent with the canonical role of SC35 as a positive regulator of splicing. However, destroying the SF2/ASF binding site promotes the inclusion of exon 11 even under damaged conditions, indicating a negative role for this protein. To further understand the role of SF2/ASF, we performed immunodepletion of SF2/ASF in normal and damaged nuclear extracts using our wild-type MDM2 construct. Our results show that depleting SF2/ASF leads to the inclusion of exon 11 under damaged conditions, a pattern similar to the SF2/ASF site mutant. We are currently assaying the effects of SC35 depletion on the splicing of the minigene. Overall, our results will provide an insight into the regulation of MDM2 alternative splicing by SC35 and SF2/ASF and its importance in cancer.

Keywords: MDM2, Alternative Splicing, SC35, SF2ASF