Abstract 4506: MDM2 alternative splicing is regulated by microRNA binding to control p53 activity

Abstract

Alternative splicing of the MDM2 is an important means by which p53 is upregulated to combat the deleterious effects of genotoxic stress. One splice variant, MDM2-ALT1 , is activated in response to genotoxic stress and is comprised of only the two terminal coding exons 3 and 12 and therefore lacks a p53 binding domain. This variant has been identified in a number of human tumors, including invasive carcinoma of the breast and lung, as well as soft tissue sarcomas. Despite its pervasiveness in tumors and the therapeutic possibilities it presents, there is very little known about the regulators of that govern this alternative splicing event. Our lab has identified a specific microRNA that is expressed at high levels in normal cells but decreased under conditions of genotoxic stress and in rhabdomyosarcoma cells, both of which are characterized by the expression of the MDM2 spliced isoform. Hence, expression of the microRNA is inversely correlated with the splice variant MDM2-ALT1. Though recent studies have shown that splicing factors can be regulated through miRNA-mediated gene silencing and indirectly influence splicing choices, we were unable to identify any splicing regulators of MDM2 to be targeted by the microRNA. Interestingly, the microRNA has been reported as one of the miRNAs that is reimported to the nucleus but has not been ascribed a role in the nuclear space. We therefore wanted to test the hypothesis that the microRNA can directly bind to the MDM2 pre-mRNA to drive splicing choices in a regulated fashion. To this end, we have identified predicted binding sites for the microRNA in the regulated region of MDM2 , within and near exons 3 and 12. We have shown that the microRNA binds specifically to these regions in MDM2 in normal cells and has decreased binding after genotoxic stress. Furthermore, we have shown that overexpression of the pre-microRNA can alleviate the induction of the MDM2-ALT1 isoform in response to genotoxic stress. We propose that microRNA binding to the MDM2 pre-mRNA regulates its splicing and plays a role in the DNA damage response and the pathway to tumorigenesis. Our work implicates for the first time that a miRNA would be acting as a splicing factor, a novel function for this class of non-coding RNAs and a novel method of regulation of the p53-MDM2 axis. Furthermore, our discoveries reveal miR-regulated splicing as a possible strategy to activate p53 and sensitize cancer cells to conventional cancer therapies. Citation Format: Matias Montes, Andrew Goodwin, Dawn S. Chandler. MDM2 alternative splicing is regulated by microRNA binding to control p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4506.