Poster abstracts
Poster number 101 submitted by Matias Montes
Splicing factors SRSF1 and SRSF2 regulation of MDM2 alternative splicing is conserved in mice and humans
Matias Montes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), Daniel Comiskey (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), John McLaughlin (Center for Molecular and Human Genetics, Nationwide Childrens Hospital), Rehan Hussain (Center for Molecular and Human Genetics, Nationwide Childrens Hospital), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)
Abstract:
The oncogene MDM2 undergoes alternative splicing and one of its spliced isoforms, MDM2-ALT1, is highly expressed in several cancers such as lung carcinoma, liposarcoma or rhabdomyosarcoma, correlating with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress. Nevertheless, the way that MDM2 splicing is controlled is not entirely understood. Previously we have utilized genotoxic stress to study the induction of the MDM2-ALT1 isoform expression in a minigene system. Using this system, we have seen that the splicing factors SRSF1 and SRSF2 act antagonistically to regulate MDM2 alternative splicing in response to stress. To determine the role of the SRSF1 and SRSF2 binding sites in the context of the endogenous MDM2 gene alternative splicing, we will use genome editing techniques for both the mouse and human MDM2 genes. Our hypothesis is that the cis elements that these factors bind are conserved in both organisms, leading us to propose that the mouse model will therefore act as a model system for testing splice altering therapies.
We have developed a damage-inducible mouse minigene, Mdm2 3-11-12s that recapitulates the splicing of the human MDM2 as well as the mouse endogenous gene by excluding its intervening exon under genotoxic stress (UV-C light). In order to study the conservation of these cis elements we used SELEX-based bioinformatics prediction algorhythms in ESEfinder 3.0 and identified conserved consensus sequences for splicing regulators SRSF1 and SRSF2 in exon 11 of the mouse Mdm2 gene. By using site-directed mutagenesis we generated mutations for the predicted SRSF1 and SRSF2 binding sites. Additionally, using the CRISPR-Cas9 technology we generated specific mutations of the binding sites in exon 11 of the mouse Mdm2 gene, to assess changes in alternative splicing. In the present work we report that regulation of Mdm2 by both SRSF1 and SRSF2 is conserved in humans and mice and that targeting their binding sites might be a mechanism to change p53 activity in specific cancer types.
Keywords: MDM2-ALT1, Alternative Splicing, Gene Editing