RRM
2012 Rustbelt RNA Meeting
RRM
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Poster abstracts
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 several isoforms. MDM2-ALT1, comprised of exons 3 and 12, is observed in over 85% of rhabdomyosarcomas, is generated in cells in response to genotoxic stress and has also been shown to accelerate tumorigenesis in a mouse model, all of which indicate a role in cancer.
In order to study the alternative splicing of MDM2 we have developed an in vitro splicing system using MDM2 minigenes and normal and cisplatin-treated HeLa nuclear extracts. The MDM2 3-11-12 minigene predominantly excludes exon 11 under UV and cisplatin 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 and performed a series of mutations in their predicted binding sites.
Our in vitro data shows that disrupting the SC35 sites in exon 11 leads to exclusion of exon 11 and disrupting the SF2/ASF site promotes the inclusion of exon 11. However, in MCF7 and HeLa cells transfected with MDM2 3-11-12 minigenes, both SC35 and SF2/ASF site mutants promote the inclusion of exon 11 under normal, UV and cisplatin treatment. We confirmed the affinity of these regulatory proteins through RNA oligonucleotide pull downs using the wild-type and mutant sequences of each binding site.
Finally, we designed antisense oligonucleotides to target these splicing regulatory sites endogenously. All ASOs blocked the formation of MDM2-ALT1 and restored full-length MDM2 in MCF7 cells upon cisplatin treatment. Taken together, this indicates that these sites are important for regulating the damage-induced alternative splicing of MDM2 and offers the potential of anti-sense-based therapy as a means of corrective splicing in cancer.
Keywords: MDM2, Alternative Splicing, SF2ASF
