Poster abstracts

Poster number 107 submitted by Zac LaRocca-Stravalle

The dichotomy of MDM2 splicing regulation in response to genotoxic stress

Zac R. LaRocca-Stravalle (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital), Hannah Ackerman (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital), Andrew K. Goodwin (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital), Dawn S. Chandler (Center for Childhood Cancer, Research Institute at Nationwide Childrens Hospital)

MDM2 is a negative regulator of the tumor suppressor protein p53. Under genotoxic stress, MDM2 alternative splicing is upregulated to produce MDM2-ALT1. MDM2-ALT1 differs from its full-length form by the skipping of exons 4 though 11 and retention of the outermost coding exons 3 and 12. However, it is unclear how the skipping of these exons is regulated. We hypothesize two models of exon skipping regulation in MDM2 pre-mRNA: (1) exon skipping is independently regulated such that exons contain binding sites that regulate their own splicing within the MDM2 transcript (exon autonomous model), (2) and skipping occurs as a single event (cassette regulon model).

To test these two models, we used a damage-inducible MDM2 minigene system. Previously, we identified conserved binding sites of splicing regulatory proteins within exon 11 that induce alternative splicing under stress – an exon autonomous event. To assess splicing regulation of other intervening exons, we used MDM2 minigenes containing an exon 4 to 10 and exposed them to genotoxic stressors, UV and cisplatin, in vitro.

We show that the minigene containing exon 4 undergoes damage-inducible alternative splicing, like exon 11. However, exons 5 to 10 were not differentially spliced in response to stress, suggesting a cassette regulon model in which exons 4 and 11 include sequences important for the splicing regulon of their intervening exons. However, these minigenes excluded intronic sequences flanking their exons. Importantly, intronic sequence mutations are known to alter splicing in cancer. Thus, to further interrogate the regulon model and test the importance of adjacent intronic sequences, we created the same minigenes but with flanking intronic regions.

Our current results support the first evidence of a cassette regulon model, which has important implications on how we understand exon recognition and alternative splicing regulation, and may serve to provide insights into new therapeutic targets.

Keywords: alternative splicing, MDM2, genotoxic stress