The superoxide dismutase 1 3′UTR maintains high expression of the SOD1 gene in cancer cells: The involvement of the RNA-binding protein AUF-1

Abstract

Superoxide dismutase 1 (SOD1) is ubiquitously expressed and the predominant dismutase in the cytoplasm. Whereas transcriptional regulation of the SOD1 gene has been well characterized, posttranscriptional regulation of the gene remains largely unknown in eukaryotes. In this study, a full-length 3′UTR of the SOD1 transcript was cloned and characterized for its ability to regulate SOD1 gene expression in human cancer cells. Inclusion of the SOD1 3′UTR in the pGL3 reporter construct dramatically enhanced the reporter activity by 10- to 220-fold in various cell lines. RT-PCR analysis, however, indicated that the reporter gene mRNA levels were only modestly altered by the SOD1 3′UTR, suggesting that the SOD1 3′UTR enhances the reporter gene activity not simply by stabilizing the mRNA but primarily by promoting translation of the protein. Bioinformatics analysis showed multiple stem and loop structures of the SOD1 3′UTR, and alterations in this secondary structure led to remarkably reduced reporter gene activity. Importantly, introducing the SOD1 3′UTR into cancer cells attenuated endogenous SOD1 expression in a concentration-dependent manner, indicating the involvement of RNA trans-acting factors in this process. Using siRNA and RNA immunoprecipitation techniques, we identified AUF-1, an RNA-binding protein, as a positive regulator of SOD1 expression through its 3′UTR. Consequently, AUF-1 was found to regulate redox balance in our cell model systems. Furthermore, in human ovarian, esophageal, and pancreatic cancer tissues, the expression of SOD1 was significantly correlated with that of AUF-1, further supporting the importance of AUF-1 in regulating SOD1 gene expression.