Talk abstracts

Talk on Friday 08:10-08:30pm submitted by Ravi Singh

Alternative splicing of p53 modulators MDM2 and MDM4 in response to cytotoxic stress

Ravi K. Singh (Molecular, Cellular and Developmental Biology program MCDB, The Ohio State University, Columbus, OH.), Edward Xie (Center for Childhood Cancer, Childrens Research Institute, Columbus, OH.), Jaquelyn Bitler (Center for Childhood Cancer, Childrens Research Institute, Columbus, OH.), Dawn Chandler (Center for Childhood Cancer, Childrens Research Institute and department of pediatrics, The Ohio State University, Columbus, OH.)

Abstract:
HDM2/p53 interaction controls p53 levels in cells, which is important for cell cycle progression and tumor development. Previous work in our lab has identified unique alternatively spliced form of MDM2 and MDM4, in response to UV, and cis-platinum but not to equitoxic levels of gamma radiation. The alternatively spliced variant of MDM2 can interfere with p53/MDM2 interaction by down-regulating MDM2 and in turn can activate p53. We have shown that stress induced alternative splicing of MDM2 and MDM4 is conserved between humans and mice. We also reported that formation of alternative splicing of MDM2 and MDM4 is through a specific pathway independent of p53, ARF, MDM2 over-expression and ATM/ATR kinases. To investigate the pathway, which induces alternative splicing we constructed human MDM2 mini-genes using the minimal regions of conserved sequences between MDM2 and MDM4 and their corresponding human homologs. These constructs have the ability to alternatively splice in vivo upon UV exposure. We also performed in vitro splicing of these 32P-labeled mini-gene transcripts using HeLa nuclear extract. In vitro splicing also results in the expected spliced products including the UV-induced alternatively spliced isoform. Using this assay we can map the minimal cis-elements and trans factors needed for alternative splicing of MDM2. Posttranslational modifications and changes in sub cellular localization of RNA processing proteins have been reported after cytotoxic stress. These changes can have global impact on RNA processing and may also regulate alternative splicing of MDM2. This study places alternative splicing of p53 modulators in the damage response pathway and introduces a new role for splicing regulators in the DNA damage response.

Keywords: alternative splicing, MDM2