Poster abstracts

Poster number 118 submitted by Ravi Singh

Identification and Characterization of Factors Involved in Damage-induced Alternative Splicing of MDM2

Ravi K. Singh (The Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital and the Department of Pediatrics, The Ohio State University, Columbus, Ohio.), Dawn S. Chandler (The Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital and the Department of Pediatrics, The Ohio State University, Columbus, Ohio.)

Abstract:
Murine double-minute 2, MDM2, and its related family member MDM4 are primary regulators of protein level and transcriptional activity of the tumor suppressor protein p53. Previous work in our lab has identified alternatively spliced forms of MDM2 and MDM4, induced specifically in response to UV exposure and cisplatin treatment. Alternative splicing of MDM2 and MDM4 in response to these stressors is conserved between human and mouse.

Using damage-responsive mini-gene constructs of MDM2, we are trying to identify the minimal cis elements and trans factors that may be involved in the regulation of MDM2 splicing in response to stress. In order to identify the cis elements that may be important in binding of damage responsive factors, we performed in vitro UV cross-linking with normal and cisplatin treated nuclear extracts using RNAs spanning the mini-gene. Two regions displayed differential binding of proteins in our cross-linking assay and these regions are conserved between human and mouse suggesting these regions may have important function in the regulation of MDM2 splicing. We performed RNA affinity chromatography using biotinylated RNAs of the two regions followed by mass spectrometry. We have identified several potential factors and are currently testing their role in the regulated splicing of MDM2.

Expression of alternatively spliced forms of MDM2 and MDM4 are associated with various cancers. The factors we identified may be effectors of the damage-induced spliceome and misregulation of these factors may result in altered gene expression leading to the disease state. This study will lead to a better understanding of global signaling affecting regulation of RNA processing factors involved in generating a damage-induced spliceome.

Keywords: MDM2, Alternative splicing