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 approximately 50-fold reduction in Se-GPx1 activity and an approximately 20-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.

FIG. 1

Hepatocytes from rats fed a Se-supplemented diet for 12 weeks have 2% of the Se-GPx1 activity and 5% of the level of Se-GPx1 mRNA of hepatocytes from rats fed a Se-supplemented diet for the same amount of time. Rats were analyzed in pairs in which one was fed a Se-supplemented diet and the other was fed a Se-deficient diet, and the periods of feeding for both members of the pair were the same. Hepatocytes were isolated according to the methods of Seglen (44) and Boyer et al. (11), as modified by us (see Materials and Methods). (A) Se-GPx1 activity was measured according to the method of Paglia and Valentine (36), as modified by Reddy et al. (39). Hepatocytes from three different animals per dietary group were analyzed, and duplicate measurements were taken for each animal. Measurements are presented as means ± standard deviations. (B) Total hepatocyte RNA (25 μg) was electrophoresed in agarose, transferred to a nylon membrane, and hybridized to a 311-bp EcoRI-EcoRI fragment of Se-GPx1 cDNA (38) and a 428-bp fragment, generated by RT-PCR, that consists of the open reading frame of cytoplasmic β-actin cDNA. Lanes marked with a minus sign contain RNAs isolated from three different animals fed a Se-deficient diet. Lanes marked with a plus sign contain RNAs isolated from three different animals fed a Se-supplemented diet. The level of Se-GPx1 mRNA was normalized to the level of β-actin mRNA in order to control for variations in the amounts of RNAs loaded in lanes. The normalized value for each rat fed a Se-supplemented diet (+Se) was considered to be 100%, and the normalized value for the corresponding rat fed a Se-deficient diet was calculated as a percentage of that of the rat fed a Se-supplemented diet.

FIG. 2

The reduced level of Se-GPx1 mRNA in Se-deficient hepatocytes is due to a decrease in the level of cytoplasmic Se-GPx1 mRNA but not nuclear Se-GPx1 mRNA. Nuclear (N) and cytoplasmic (C) RNA was isolated (method 1 [4]) from hepatocytes of each pair of rats fed either a Se-deficient (−) or a Se-supplemented (+) diet. Se-GPx1 and β-actin RNAs in each sample were analyzed by RT-PCR. The left-most four lanes consist of serial dilutions of cytoplasmic RNA (5.0, 1.3, 0.6, and 0.3 μg, from left to right), which were used to establish that there is a linear relationship between the amount of input RNA and the amount of each RT-PCR product. Size standards for RT-PCR products of Se-GPx1 pre-mRNA and Se-GPx1 mRNA were provided by the PCR amplification of, respectively, 25 pg of pmCMV-GPx1 (Se-GPx1 gene) and 25 pg of pGPx1211 (Se-GPx1 cDNA) with the same primers that were used to amplify hepatocyte cDNA.

FIG. 3

Incubating either L cells or NIH 3T3 cells in Se-deficient medium for 24, 48, or 72 h decreases Se-GPx1 activity to 50 to 55% of the activity of Se-supplemented cells. Activity was measured according to the method of Paglia and Valentine (36), as modified by Reddy et al. (39). Each cell line was mock transfected with either DEAE dextran (L cells) or calcium phosphate (NIH 3T3 cells) and incubated for an additional 12 h before the medium was changed to either Se-deficient (−) or Se-supplemented (+) medium. Se-GPx1 activities are presented as the means of duplicate measurements for each time point.

FIG. 4

(A) Structures of the mCMV-GPx1 gene and derivative alleles. The shaded box represents the 550-bp XbaI-EcoRI fragment that harbors the mCMV promoter. The open boxes represent exons, the intervening line represents the single 216-bp intron, and the right-most line represents 3′ flanking DNA. ATG(0), TGA(46), and TAA(201) represent, respectively, the initiation codon, Sec codon, and termination codon. Mutations that convert the Sec codon to either a Cys codon (TGC) or a premature termination codon (TAA) are indicated below the gene structure. (B) The level of mCMV-GPx1 mRNA in total RNA of NIH 3T3 cells grown in Se-deficient medium (−) is 50% of the level in NIH 3T3 cells grown in Se-supplemented (+) medium. Cells were either untransfected or transiently transfected with pmCMV-GPx1 (25 μg) and the reference plasmid pmCMV-G1 (25 μg). Total RNA was isolated, and RT-PCR was used to quantitate mCMV-GPx1 and mCMV-G1 mRNAs. The left-most five lanes contain twofold serial dilutions of RNA from Se-supplemented cells in order to demonstrate a linear relationship between the amounts of input RNA and RT-PCR products. The level of mCMV-GPx1 mRNA was normalized to the level of mCMV-G1 mRNA. The normalized value for mCMV-GPx1 mRNA in Se-deficient cells was then calculated as a percentage of the normalized value for mCMV-GPx1 mRNA in Se-supplemented cells, which was considered to be 100%. The values from two independently performed experiments did not differ by more than 3%. (C) The ratio of cytoplasmic mCMV-GPx1 mRNA to nuclear mCMV-GPx1 mRNA is decreased in Se-deficient NIH 3T3 cells by a decay pathway that is dependent on recognition of the Sec codon as a termination codon. Transfections and analyses of RNA were as described in the legend to Fig. 4B, except that nuclear and cytoplasmic RNAs were purified and analyzed as described in the legend to Fig. 2. The left-most four lanes contain twofold dilutions of cytoplasmic RNA from Se-supplemented NIH 3T3 cells transiently transfected with pmCMV-GPx1-TGC(46). GPx1, GPx1-TAA(46), and GPx1-TGC(46) signify pmCMV-GPx1 plasmids harboring at position 46 the wild-type sequence (TGA), a nonsense codon (TAA), and a Cys codon (TGC), respectively. The two right-most lanes, which reflect PCR analyses of intronless pmCMV-GPx1 DNA and pmCMV-GPx1 DNA, provide molecular weight standards for pre-mRNA and mRNA, respectively.