Abstract
Alternative splicing of the oncogene MDM2 is a phenomenon that occurs in cells in response to genotoxic stress and is also a hallmark of several cancer types with important implications in carcinogenesis. However, the mechanisms regulating this splicing event remain unclear. Previously, we uncovered the importance of intron 11 in MDM2 that affects the splicing of a damage-responsive MDM2 minigene. Here, we have identified discrete cis regulatory elements within intron 11 and report the binding of FUBP1 (Far Upstream element-Binding Protein 1) to these elements and the role it plays in MDM2 splicing. Best known for its oncogenic role as a transcription factor in the context of c-MYC, FUBP1 was recently described as a splicing regulator with splicing repressive functions. In the case of MDM2, we describe FUBP1 as a positive splicing regulatory factor. We observed that blocking the function of FUBP1 in in vitro splicing reactions caused a decrease in splicing efficiency of the introns of the MDM2 minigene. Moreover, knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions. These results indicate that FUBP1 is also a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1.
Highlights
MDM2 is alternatively spliced into shorter isoforms under DNA damage and in several cancers through unknown mechanisms
Using this minigene we showed that intron 11 of MDM2 contains conserved elements that are important for its stress-induced alternative splicing [22]
We hypothesized that the differential splicing of the MDM2 minigene in nuclear extracts from normal and damage-treated cells is the result of differential binding of trans regulatory splicing factors to cis regulatory elements within the conserved region of intron 11, and in this study we endeavored to define these elements and the proteins that bind them
Summary
MDM2 is alternatively spliced into shorter isoforms under DNA damage and in several cancers through unknown mechanisms. Knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions These results indicate that FUBP1 is a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1. Treatment with DNA-damaging agents such as UV irradiation and cisplatinum induces the formation of MDM2-ALT1 transcripts [20, 21] We have utilized this inducible system to develop an in vitro splicing assay using nuclear extracts from normal and cisplatinum-treated HeLa S3 cells and a damageresponsive MDM2 minigene. We show here the binding of endogenous FUBP1 to intronic splicing enhancer elements in intron 11 of MDM2 and the role it plays in enhancing the efficient splicing of full-length MDM2, making this the second report to identify a splicing regulatory role for FUBP1 and the first report to describe an enhancer role for this splicing regulator
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