Poster abstracts

Poster number 29 submitted by Matias Montes

MDM2 alternative splicing is regulated by microRNA binding: A novel pathway of MDM2 regulation

Matias Montes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)

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, there is very little known about the regulators of that govern this alternative splicing event.
Our lab has identified expression of a specific microRNA to be inversely correlated with the splice variant MDM2-ALT1. Hence, the microRNA 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. 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.

Keywords: miRNA, MDM2, Alternative Splicing