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
, 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
) 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 studies of Escherichia coli
(reviewed in reference 10
), (ii) the discovery of mammalian homologs to some of the bacterial factors that mediate the cotranslational incorporation of Sec (27
), and (iii) information on mammalian mRNA sequences that mediate the incorporation of Sec (7
) have contributed to elucidating the mechanism by which a UGA codon is recognized as a Sec codon. In mammals, recognition requires selenium (Se), a metabolic pathway that converts Se to selenocysteyl-tRNA[Ser]Sec
, and at least one cis
-acting Sec insertion sequence element that presumably associates with the specialized elongation factor. Studies of the type I iodothyronine deiodinase (5′ DI) gene, in which the single TGA codon was converted to a cysteine (Cys) codon, indicate that cells transiently overexpressing the gene are able to produce 20- to 400-fold-more protein from the Cys-containing allele than from the TGA-containing allele (8
). Therefore, at least in transiently transfected cells, Sec is incorporated at the UGA codon of 5′ DI mRNA only inefficiently, as may be the case for other selenoprotein mRNAs.
Consistent with the concept that UGA-containing selenoprotein mRNAs can be reduced in abundance when the UGA codon(s) is recognized as nonsense, Se deprivation reduces the abundance of certain selenoprotein mRNAs, some more effectively than others. For example, rats or mice fed a Se-deficient diet for 42 to 135 days manifest an 80 to 95% drop in the level of Se-dependent glutathione peroxidase 1 (Se-GPx1) mRNA in liver (28
) that is not attributable to a decrease in Se-GPx1 gene transcription (6
). The level of endogenous Se-GPx1 mRNA is also decreased in Se-deficient cultured cells, although not by more than 70%, even if the cells derive from liver (1
). Se deficiency reduces the level of other selenoprotein mRNAs, including those for 5′ DI (23
), selenoprotein P (23
), and selenoprotein W (47
). However, Se deficiency does not reduce the level of all selenoprotein mRNAs, as exemplified by mRNA for phospholipid hydroperoxide glutathione peroxidase (PHGPx) (5
). Consistent with Se having different effects on different mRNAs, the results of incubating H4 rat hepatoma cells with actinomycin D indicate that Se deficiency decreases the half-life of total-cell Se-GPx1 mRNA but is of no consequence to the half-life of cytoplasmic PHGPx mRNA (5
). It is possible that Se regulates the level of selenoprotein mRNAs not only in translation-dependent mechanisms but also in translation-independent mechanisms, and this possibility has been proposed given that (i) feeding Se to Se-deficient rats results in an increase in the level of Se-GPx1 mRNA before a detectable increase in Se-GPx1 activity (45
) and (ii) the level of Se-GPx1 activity in rats fed different amounts of Se does not always parallel the level of Se-GPx1 mRNA (6
). The mechanism by which Se deprivation reduces the expression of Se-GPx1 cDNA in Chinese hamster ovary (CHO) cells has been demonstrated to be dependent on sequences within the 3′ untranslated region (49
), which may also be consistent with a translation-independent mechanism.
In order to clarify if Se (i) regulates the level of Se-GPx1 mRNA via the Sec codon, (ii) affects Se-GPx1 gene expression independently of the Sec codon, or (iii) does both, Se deficiency was induced in rat liver and in NIH 3T3 cells transiently transfected with one of several Se-GPx1 alleles that harbored different sequences at their Sec codons. Results indicate that Se deficiency reduces the abundance of cytoplasmic Se-GPx1 mRNA without altering the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. Regardless of the Se concentration, changing the Sec codon from TGA to a TAA nonsense codon decreased the ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA while changing the Sec codon to a TGC cysteine codon increased the ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA. Neither change altered the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. These data indicate that Se deprivation regulates the abundance of cytoplasmic Se-GPx1 mRNA, presumably by eliciting the nonsense-codon-mediated decay of cytoplasmic mRNA.