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Mol Cell Biol, May 1998, p. 2932-2939, Vol. 18, No. 5
Department of Human Genetics, Roswell Park
Cancer Institute, Buffalo, New York 14263,1
and
Department of Veterinary Science, Pennsylvania State
University, University Park, Pennsylvania 168022
Received 14 November 1997/Returned for modification 23 December
1997/Accepted 17 February 1998
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 ~50-fold reduction in Se-GPx1
activity and an ~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.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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
*
Corresponding author. Mailing address: Department of
Human Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-3325. Fax: (716) 845-8449. E-mail:
Maquat{at}sc3101.med.buffalo.edu.
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