Poster abstracts
Poster number 84 submitted by Zac LaRocca-Stravalle
The Dichotomy of MDM2 Splicing Regulation under Genotoxic Stress
Zac R. LaRocca-Stravalle (Nationwide Childrens Hospital, Center for Childhood Cancer), Andy K. Goodwin (Nationwide Childrens Hospital, Center for Childhood Cancer), Hannah R. Ackerman (Nationwide Childrens Hospital, Center for Childhood Cancer), Dawn S. Chandler (Nationwide Childrens Hospital, Center for Childhood Cancer)
Abstract:
MDM2 is overexpressed in sarcomas and is a negative regulator of the tumor suppressor protein p53 [1]. Under genotoxic stress, MDM2 undergoes alternative splicing to produce MDM2-ALT1, which lacks the p53 binding domain [2]. This isoform retains the outermost coding exons 3 and 12 while exons 4 though 11 are skipped. 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 transcript (exon autonomous model), and (2) skipping occurs as a single event (cassette regulon model) wherein exons 4 and 11 get spliced together.
To test these models, we used a damage-inducible MDM2 minigene system with ALT1 exons 3 and 12 and a middle intervening exon 4 to 11 (3-X-12). Previously, we showed that when exposed to genotoxic stressors, UV and cisplatin, in vitro, minigenes containing 4 and 11 were damage inducible but exons 5 to 10 were not. Additionally, we identified conserved binding sites of splicing regulatory proteins within exon 11 that induce alternative splicing under stress [3]. Thus, we hypothesize a regulon model of exon skipping.
Here, we expand on this work by adding longer intronic sequences corresponding to the native exons to further interrogate their roles in splicing, as intronic regions contain important splicing regulatory elements that are known to alter splicing in cancer. We show that exons 4 and 11 remain damage responsive, in addition to exon 5. Exons 6, 8, 9 and 10 exhibit noticeable yet non-significant damage responsive induction.
Our current results suggest MDM2 exon skipping regulation may occur as exon autonomous events. Nonetheless, because the distal exons exhibit the greatest damage response, and exon 11 itself has been shown to regulate splicing of the preceding 8 exons, a regulon splicing event is also supported in MDM2. Thus, there may exist a hybrid combination of exon skipping, such that exon splicing can occur both autonomously and as a regulon. This work may serve to provide insights into new splice-targeting therapeutics involving the removal of multiple exons.
References:
[1] Sciot R. (2021). MDM2 Amplified Sarcomas: A Literature Review. Diagnostics (Basel, Switzerland), 11(3), 496. https://doi.org/10.3390/diagnostics11030496
[2] Jacob, A. G., O'Brien, D., Singh, R. K., Comiskey, D. F., Jr, Littleton, R. M., Mohammad, F., Gladman, J. T., Widmann, M. C., Jeyaraj, S. C., Bolinger, C., Anderson, J. R., Barkauskas, D. A., Boris-Lawrie, K., & Chandler, D. S. (2013). Stress-induced isoforms of MDM2 and MDM4 correlate with high-grade disease and an altered splicing network in pediatric rhabdomyosarcoma. Neoplasia (New York, N.Y.), 15(9), 1049–1063. https://doi.org/10.1593/neo.13286
[3] Comiskey, D. F., Jr, Jacob, A. G., Singh, R. K., Tapia-Santos, A. S., & Chandler, D. S. (2015). Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage. Nucleic acids research, 43(8), 4202–4218. https://doi.org/10.1093/nar/gkv223
Keywords: Alternative Splicing , Cancer, MDM2