Poster abstracts
Poster number 28 submitted by Zac LaRocca-Stravalle
The Dichotomy of Splicing Regulation Regulation under Genotoxic Stress
Zac LaRocca-Stravalle (Nationwide Childrens), Andy K. Goodwin (Nationwide Childrens), Hannah R. Ackerman (Nationwide Childrens), Dawn S. Chandler (Nationwide Childrens)
Abstract:
MDM2 is a negative regulator of the tumor suppressor protein p53. Under genotoxic stress, MDM2 undergoes alternative splicing to produce MDM2-ALT1, among other isoforms. 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 alternative splicing of these exons is regulated. We propose 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), and (2) skipping occurs as a single event (cassette regulon model) due to its secondary structure wherein exons 4 and 11 get spliced together.
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. To assess splicing regulation of other intervening exons, we used MDM2 minigenes containing an exon 4 to 10 (3-X-12) and exposed them to genotoxic stressors, UV and cisplatin, in vitro. Based on the autonomous skipping of exon 11 minigene (3-11-12), we hypothesize that other MDM2 exons are involved in autonomous skipping events.
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 do not include flanking intronic sequences that correspond to their respective 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 incorporated flanking intronic regions into the same minigenes.
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: MDM2 , Alternative Splicing, Cancer