Poster abstracts
Poster number 71 submitted by Matias Montes
Elucidating the mechanisms of regulation for MDM2 alternative splicing
Matias Montes (Center for Childhood Cancer and Blood diseases Nationwide Childrens Hospital), Dawn Chandler (Center for Childhood Cancer and Blood diseases Nationwide Childrens Hospital)
Abstract:
MDM2, a well-known negative regulator of the tumor suppressor protein p53 undergoes complex alternative splicing. One of its spliced isoforms, MDM2-ALT1 comprised of exons 1 through 3 and 12, is highly expressed in several cancers such as lung carcinoma, liposarcoma and rhabdomyosarcoma (RMS). In RMS, MDM2-ALT1 expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress. The way that MDM2 splicing is controlled is not well understood. Here we explore the possibility that a nuclear miRNA, miR29b, interplays with RNA binding proteins to regulate MDM2 splicing.
miR-29b is part of the miR-29 family, and has been reported as one of the miRNAs that localize to the nuclear compartment of cells. However, its function there is still unknown. Additionally, miR-29b has been shown to act as a tumor suppressor. In RMS, we have shown how miR-29b levels are downregulated in both cell lines as well as in patient samples. Here, we show how miR-29b is downregulated after genotoxic stress, which inversely correlates with MDM2-ALT1 expression, similarly to RMS. Furthermore, we show how miR-29b influences the alternative splicing of MDM2, which is not through canonical gene-silencing of splicing factors. We have identified predicted binding sites for the miR-29b in the regulated region of MDM2 pre-mRNA. Moreover, we demonstrate that miR-29b directly binds to the MDM2 pre-mRNA and that the site present in the exon 12 is important for alternative splicing regulation. Finally, we show how Argonaute 1 antagonizes miR-29b influence over the MDM2 alternative splicing, and interaction between AGO1 and miR-29b is increased after genotoxic stress. In summary, in this work we report for the very first time how a nuclear miRNA has direct involvement regulating the alternative splicing of a gene.
Keywords: alternative splicing, miRNAs, MDM2