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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.

SUBMITTER: Zhang S

PROVIDER: S-EPMC4508224 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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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.

Zhang Shuyu S Xue Jing J Zheng Jie J Wang Shuai S Zhou Jundong J Jiao Yang Y Geng Yangyang Y Wu Jinchang J Hannafon Bethany N BN Ding Wei-Qun WQ

Free radical biology & medicine 20150420

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 en ...[more]

PMID: 25908445

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Project description:Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.

Dataset Information

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.

Publications

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.

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