Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA

Abstract

The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an ϳ50-fold reduction in Se-GPx1 activity and an ϳ20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.Many organisms, including bacteria, Saccharomyces cerevisiae, and vertebrates, appear to have established mechanisms that eliminate the production of mRNAs that prematurely terminate translation because of a frameshift or nonsense mutation (reviewed in references 32, 33, 37, and 42). Therefore, the discovery of mRNAs in both bacterial and mammalian cells in which one or more UGA codons are purposefully used as selenocysteine (Sec) codons rather than as nonsense codons (reviewed in references 10 and 30) raises the interesting issue of whether these mRNAs are reduced in abundance when the UGA codon is recognized as nonsense.Cues from (i) biochemical and genetic stu...

Selenoprotein expression is regulated at multiple levels in prostate cells

“…Figure 4C shows that GPX2 levels were not detectable in PC-3 or LNCaP cells, even in the presence of added selenium. In addition, and consistent with GPX1, selenium supple- npg This is consistent with the protein levels observed in Figure 4A and the previous reports showing that GPX1 mRNA is unstable when selenium is deficient [21][22][23][24]. GPX4 mRNA levels are not as consistent with protein levels since clear expression is detectable even in PC-3 cells under low selenium conditions where GPX4 protein is lacking, indicating a clear case of regulation at the translational level.…”

“…This is also true for GPX1, but to a lesser extent, because mRNA levels in the absence of selenium supplementation are reduced. This is very likely to be the result of lower mRNA stability as has been observed for GPX1 in multiple studies [21][22][23][24]. In the case of GPX2, the expression observed appears to be dramatically regulated at the RNA level, most likely at the level of transcription because its mRNA is not detectable even in the presence of supplemental selenium.…”

Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise

“…Putative NMD-inducing features included upstream open reading frames (uORFs; 70 transcripts), alternative splicing that introduces nonsense codons or frameshifts (21 transcripts, some of which undergo alternative splicing specifically in HeLa cells 12,13 ) and introns in the 3¢ untranslated region (UTR; 9 transcripts). Two transcripts encoding proteins containing selenocysteine were also upregulated, consistent with the alternative recognition of the UGA selenocysteine codon as a signal for translation termination 14 . Additionally, we observed upregulation of nonfunctional transcripts derived from mariner 2 transposon (HSMAR2) remnants and human endogenous retrovirus H (HERV-H) sequences that have acquired premature termination codons (PTCs) during evolution.…”

mRNA surveillance in mammalian cells: The relationship between introns and translation termination

“…The idea that an intronless gene generates mRNA that is subject to NMD has also been negated by our studies demonstrating that expression of an intronless gene for glutathione peroxidase (GPx) 1 under conditions of selenium deficiency fails to increase the efficiency of NMD despite increasing the efficiency with which the Sec (TGA) codon is recognized as nonsense (Moriarty et al+, 1997)+ Furthermore, converting the Sec codon to a TAA nonsense codon in the context of an intronless GPx1 allele does not change the level of GPx1 mRNA, despite the fact that the TAA codon is recognized as nonsense more efficiently than the Sec codon, regardless of the Se concentration (Moriarty et al+, 1997)+ In contrast, expression of the full-length GPx1 gene under conditions of Se deficiency or expression of the full-length GPx1 gene harboring a TAA nonsense codon in place of the Sec codon does increase the efficiency of NMD, as would be expected considering that the sole intron is situated 105 bp (i+e+, more than 50-55 bp) downstream of the Sec codon (Moriarty et al+, 1997(Moriarty et al+, , 1998)+…”

“…In mammals, as in all organisms examined, mRNAs that prematurely terminate translation are abnormally reduced in abundance by a mechanism called nonsense-mediated mRNA decay (NMD) or mRNA surveillance (for reviews, see Maquat, 1995Maquat, , 1996Li & Wilkinson, 1998;Culbertson, 1999;Hentze & Kulozik, 1999;Hilleren & Parker, 1999)+ This mechanism is thought to have evolved to eliminate nonsense-containing RNAs that arise as a consequence of (1) mutations in germ-line or somatic DNA or (2) routine abnormalities in gene expression due to abnormalities in, for example, transcription initiation, splicing, and somatic rearrangements of the type that characterize the immunoglobulin and T-cell receptor genes+ The elimination of nonsense-containing mRNAs protects cells from the potentially deleterious effects of the encoded truncated proteins, which can manifest new or dominant-negative functions (Kazazian et al+, 1992;Pulak & Anderson, 1993;Hall & Thein, 1994;Cali & Anderson, 1998)+ In addition to eliminating abnormal transcripts, NMD also regulates the expression of certain mRNAs that are not abnormal+ Examples in mammalian cells are provided by certain selenoprotein mRNAs that terminate translation at a UGA codon for the inefficiently incorporated amino acid selenocysteine (Sec; Moriarty et al+, 1997Moriarty et al+, , 1998+ Other examples will undoubtedly resemble natural substrates found in other organisms such as the alternatively spliced mRNAs of Caenorhabditis elegans that retain an internal exon harboring an in-frame termination codon (Morrison et al+, 1997), and transcripts of Saccharomyces cerevisiae that contain a small open reading frame upstream of the primary open reading frame (Leeds et al+, 1992;Pierrat et al+, 1993)+ Based on studies of disease-associated and in vitromutagenized genes, a rule for termination-codon position within intron-containing genes has been established for mammalian cells: only those termination codons located more than 50-55 nt upstream of the 39-most exon-exon junction mediate mRNA decay (Cheng et al+, 1994;Thermann et al+, 1998;Zhang et al+, 1998aZhang et al+, , 1998b)+ In support of this rule, more than 98% of genes having one or more 39-untranslated exons terminate translation less than 50 nt upstream of the 39-most exon-exon junction (Nagy & Maquat, 1998)+ There is a growing consensus that exon-exon junctions function in NMD+ According to current thinking, the process of pre-mRNA splicing influences product mRNP structure and, in so doing, NMD, by leaving a mark at the junctions ...…”