Abstract 3080: Novel splicing activity for RNA binding protein CUGBP2 in radiation-induced mitotic catastrophe

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL RNA binding protein CUGBP2 encodes various functions at the posttranscriptional level including alternative splicing (e.g. tau, troponin T), RNA editing (apolipoprotein B) and mRNA translation (cyclooxygenase-2, Mcl1). CUGBP2 contains two RNA recognition motifs (RRMs) near the N terminus, a divergent domain loaded with alanine and glutamine residues, and a third RRM near the C terminus. Based on deletion analyses, the divergent domain is believed to encode activation modules necessary for splicing activity. Recently, we have demonstrated that there are three promoters that drive the expression of the CUGBP2 gene, transcription from which results in three variant forms of the protein. Translation of the transcripts driven from the upstream promoters adds additional amino acids to the N-terminus of CUGBP2. In mouse intestine, the predominant form of the protein is variant 2. However, after exposure to 6 Gy γ-irradiation (IR), the levels of CUGBP2 mRNA shift to being predominantly variant 1. In colon cancer cells, IR induced the cells to undergo mitotic catastrophe, a poorly understood form of cell death where cells escape the G2/M checkpoint, enter mitosis prematurely and concurrently induce an apoptotic pathway. Interestingly, there was an increase in the CUGBP2 variant that is expressed from the proximal promoter (var 1). Furthermore, downregulation of CUGBP2 through specific siRNA significantly reduced the IR-induced mitotic catastrophe. While var 1 predominantly localizes to the nucleus, the other isoforms primarily reside in the cytoplasm. We next sought to determine the mechanism by which CUGBP2 induces mitotic catastrophe. We have observed that it occurs in part through inhibiting the translation of transcripts encoding anti-apoptotic proteins Bcl-2 and Mcl1. CUGBP2 also affected the splicing of two transcripts that encode microtubule-binding proteins. MARK2 (microtubule affinity regulating kinase 2) is involved in the breakdown of the microtubule network, which is important for cell division and CKAP5 (cytoskeleton associated protein 5) is a protein present in the microtubule-microfilament complex. Exons 15 and 36 are alternatively spliced in MARK2 and CKAP5, respectively. Using RT-PCR analyses, we observed that following CUGBP2 var 1 overexpression, these two exons, 15 and 36 were spliced out from the MARK2 and CKAP5 mRNA, respectively. This was associated with the cells undergoing radiation-induced mitotic catastrophe. In summary, CUGBP2 has been shown to undergo alternative promoter usage and alternative use of the first exon following exposure of cells to IR. With the increase in var 1, we observe inhibition of Bcl2 and Mcl1 translation, and increased alternative splicing of MARK2 and CKAP5. These data, taken together, suggest that CUGBP2 regulates cell viability by two different posttranscriptional mechanisms of alternative splicing and translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3080. doi:10.1158/1538-7445.AM2011-3080