Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels

Abstract

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 6 2% and 15 6 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 39-UTR can completely replace the GPX1 39-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 39-UTR to b-globin mRNA can convey significant Se regulation to b-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 39-UTR and a Sec codon followed by an intron.

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

“…Nevertheless, studies in addition to those reported here offer no evidence that the PHGPx SECIS confers immunity to NMD in cultured cells: hybrid genes in which the 3Ј UTR of GPx1 mRNA was substituted for that of PHGPx mRNA generated mRNA susceptible to NMD under either Se-adequate or Se-deficient conditions (Weiss and Sunde, 1998). Furthermore, it is difficult to conceive of a SECIS element so effective that it would function with comparable efficiency regardless of the Se concentration.…”

“…Second, at least for the PHGPx gene of pig, which is the only characterized PHGPx gene, the sole Sec codon resides within exon 3 and is followed by four introns, all of which reside Ͼ50 -55 bp downstream of the Sec codon (Brigelius-Flohé et al, 1994). In fact, the distance between the Sec codon and the closest downstream intron for both PHGPx and GPx1 genes is 105 bp, and this intron has been shown to be critical for the NMD of GPx1 mRNA (Moriarty et al, 1997;Weiss and Sunde, 1998;Sun et al, 2000). Third, based on studies of GPx1 mRNA translation (Moriarty et al, 1998), there is no reason to think that the cotranslational insertion of Sec into PHGPx protein would not be in competition with translation termination and, therefore, NMD.…”

Nonsense-mediated decay factors are involved in the regulation of selenoprotein mRNA levels during selenium deficiency

“…A recent study posits that under Se deficiency, eIF4A3, a core component of the EJC, is up-regulated and binds the SECIS element of GPx1 but not that of GPx4, competing for binding of the Sec-incorporation factor SBP2 and ultimately inhibiting Sec incorporation (Budiman et al 2009). Although this is a plausible theory, it conflicts with a previous study (Weiss and Sunde 1998), showing that the GPx4 3 ′ UTR is not able to stabilize the Se responsiveness of GPx1 mRNA. Another explanation could relate to the relative abundance of each of two isoforms of the Sec-tRNA.…”

Selenoproteins: Molecular Pathways and Physiological Roles

“…This "hierarchical" regulation of selenoprotein expression occurs at both mRNA abundance and translational levels; however, the underlying mechanisms are not fully understood (335). The decreased mRNA abundance of stress-related selenoproteins under conditions of Se deficiency was attributed to altered rates of mRNA turnover due to nonsense-mediated decay (NMD) rather than transcriptional regulation (260,366). In eukaryotes, NMD pathway is used to eliminate mRNA transcripts with premature stop codons.…”