Molecular and Cellular Biology, June 2001, p. 3840-3852, Vol. 21, No. 11
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.11.3840-3852.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208921; Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt2; Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 685883; Thyroid Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 0211154; Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 372325; Department of Nutrition, Division of Nutritional Science, Cornell University, Ithaca, New York 148536; Science Applications International Corporation, Frederick Cancer Research and Development Center, Frederick, Maryland 217027; Radiation Oncology Center, Washington University, St. Louis, Missouri 631108; Department of Human Nutrition, University of Illinois at Chicago, Chicago, Illinois 606129; and Laboratory of Molecular Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea10
Received 23 January 2001/Returned for modification 12 March 2001/Accepted 20 March 2001
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ABSTRACT |
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Selenocysteine (Sec) tRNA (tRNA[Ser]Sec) serves as
both the site of Sec biosynthesis and the adapter molecule for donation
of this amino acid to protein. The consequences on selenoprotein
biosynthesis of overexpressing either the wild type or a mutant
tRNA[Ser]Sec lacking the modified base,
isopentenyladenosine, in its anticodon loop were examined by
introducing multiple copies of the corresponding tRNA[Ser]Sec genes into the mouse genome. Overexpression
of wild-type tRNA[Ser]Sec did not affect selenoprotein
synthesis. In contrast, the levels of numerous selenoproteins decreased
in mice expressing isopentenyladenosine-deficient (i6A
) tRNA[Ser]Sec in a
protein- and tissue-specific manner. Cytosolic glutathione peroxidase
and mitochondrial thioredoxin reductase 3 were the most and least
affected selenoproteins, while selenoprotein expression was most and
least affected in the liver and testes, respectively. The defect in
selenoprotein expression occurred at translation, since selenoprotein
mRNA levels were largely unaffected. Analysis of the
tRNA[Ser]Sec population showed that expression of
i6A
tRNA[Ser]Sec altered the
distribution of the two major isoforms, whereby the maturation of
tRNA[Ser]Sec by methylation of the nucleoside in the
wobble position was repressed. The data suggest that the levels of
i6A
tRNA[Ser]Sec and wild-type
tRNA[Ser]Sec are regulated independently and that the
amount of wild-type tRNA[Ser]Sec is determined, at least
in part, by a feedback mechanism governed by the level of the
tRNA[Ser]Sec population. This study marks the first
example of transgenic mice engineered to contain functional tRNA
transgenes and suggests that i6A
tRNA[Ser]Sec transgenic mice will be useful in assessing
the biological roles of selenoproteins.
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INTRODUCTION |
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Selenocysteine (Sec) is encoded by UGA in selenoprotein mRNAs, making Sec the 21st naturally occurring amino acid in protein (reviewed in references 6, 19, and 35). The usage of UGA as a Sec codon represents the only addition to the genetic code since the code was deciphered in the mid-1960s. Decoding of UGA as Sec, rather than termination, requires specific secondary structures in selenoprotein mRNAs, termed Sec insertion sequences or SECIS elements, several trans-acting factors, and a unique tRNA with an anticodon complementary to UGA. The tRNA is first aminoacylated with serine, which serves as the backbone for the biosynthesis of Sec. Sec tRNA is therefore designated tRNA[Ser]Sec. It is not recognized by the standard elongation factor, eEFIA, but by a specialized factor, designated eEFsec, which exhibits specificity for both the unique tRNA structure and the amino acid (16, 46). Recruitment of the Sec-tRNA-EFsec complex to the ribosome occurs via its interaction with the SECIS binding protein 2, a protein exhibiting specificity for the SECIS elements in selenoprotein mRNAs (12).
Selenoproteins typically contain only one Sec residue per polypeptide and are expressed at relatively low levels compared to most other cellular proteins. There are fewer than 10 known prokaryotic and 20 known eukaryotic selenoproteins, but they provide a selective advantage to some organisms and are essential to others (reviewed in reference 23). The central component for the synthesis of the entire class of selenoproteins is tRNA[Ser]Sec. Thus, manipulation of the gene for this tRNA provides a potential target for better understanding the biological roles of this class of proteins. tRNA[Ser]Sec gene knockout mice, however, are embryonic lethal (7). Although this observation demonstrates an essentiality of selenoprotein expression in mammals, this lethality also precludes utilization of these mice as a tool for studying selenoproteins. The expression of genetically altered tRNA[Ser]Sec genes in transgenic mice offers an alternative approach to specifically perturb selenoprotein biosynthesis and study the biological roles of selenoproteins.
In higher vertebrates, selenoprotein expression is dictated by two
major isoacceptors that differ from each other by a single 2'-O-methyl group on the ribosyl moiety of the modified
residue, methylcarboxymethyl-5'-uridine (mcm5U), at
position 34 (reviewed in references 19 and 23). Transfer RNA[Ser]Sec methylation at position 34 results in the
formation of
methylcarboxymethyl-5'-uridine-2'-O-methylribose (mcm5Um). The methylation step is responsive to selenium
availability (9, 15, 22) and results in a conformational
change in tRNA[Ser]Sec (15). The
unmethylated form, mcm5U, is therefore the precursor of the
methylated form, mcm5Um (10, 30). In mammalian
cells and tissues, selenium deficiency is associated with a shift in
the distribution of the two isoacceptors towards the mcm5U
isoform, while selenium supplementation typically results in a shift
towards the mcm5Um isoform (9, 15, 22).
tRNA[Ser]Sec also contains additional modified residues,
including isopentenyladenosine (i6A) at position 37, pseudouridine (
) at position 55, and 1-methyladenosine at position
58 (14). The methylation of mcm5U to form
mcm5Um does not occur if i6A is not present in
the tRNA (30).
Many tRNAs that translate codons with U in their 5' position contain
i6A at position 37 (4, 5, 13). The absence of
this modification dramatically reduces the efficiency of the altered
tRNA in decoding nonsense codons in bacteria and yeast, and its
presence apparently restricts wobble and prevents misreading (4,
5, 13). Chinese hamster ovary (CHO) cells transiently
transfected with an i6A
tRNA[Ser]Sec gene showed marginal inhibition of
endogenous selenoprotein synthesis but about 80% inhibition of
selenoprotein type 1 deiodinase synthesized by cotransfection of an
expression construct encoding the gene for this selenoprotein
(47). In this same study, CHO cells treated with
lovastatin, an inhibitor of the rate-limiting step in the biosynthesis
of i6A, resulted in the inhibition of both general
selenoprotein synthesis and type 1 deiodinase. Since all other
i6A-containing tRNAs would also be expected to lack this
modified base, the use of lovastatin did not distinguish between the
possible effects of i6A
tRNA[Ser]Sec and other i6A-lacking tRNAs on
selenoprotein synthesis.
In the present study, transgenic mice containing from 2 to 20 wild-type
tRNA[Ser]Sec transgenes or from 2 to 40 i6A
tRNA[Ser]Sec transgenes
were generated, and the effects of overexpression of wild-type
tRNA[Ser]Sec and expression of the mutant
tRNA[Ser]Sec on tRNA maturation and selenoprotein
synthesis in several tissues was examined. The results of these studies
are described herein.
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MATERIALS AND METHODS |
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Materials.
[75Se]selenious acid (specific
activity, 1,000 Ci/mmol) was obtained from the Research Reactor
Facility, University of Missouri (Columbia, Mo.),
[
-32P]dCTP and [
-32P]ATP (specific
activities, ~6,000 Ci/mmol each) were obtained from New England
Nuclear, [3H]serine (specific activity, 36 Ci/mmol) and
Hybond-N+ nylon membranes were obtained from Amersham,
polynucleotide kinase and reverse transcriptase were obtained from
Boehringer Mannheim, the RNeasy kit was obtained from Qiagen, human
-actin cDNA probe was obtained from Clontech, and NACS PREPAC ion
exchange columns, restriction endonucleases, and agarose were obtained
from Gibco-BRL. All other reagents were commercial products of the
highest grade available. Inbred FVB/N mice were obtained from Charles
River (Frederick, Md.), and B6SJL hybrid mice were from Jackson
Laboratories, Bar Harbor, Maine. The care of animals was in accordance
with the National Institutes of Health institutional guidelines under the expert direction of G. Lidl (National Cancer Institute, NIH, Bethesda, Md.).
Transgenic mice and excision of tissues and organs.
A
2.17-kb StuI-PvuII fragment containing 1.93 kb of
mouse DNA encoding the wild-type tRNA[Ser]Sec gene
(41) and 0.24 kb of pBluescript II vector DNA was used for
developing a colony of B6SJL hybrid transgenic mice carrying the
wild-type transgene at the National Institute of Child Health and Human
Development Transgenic Mouse Development Facility, University of
Alabama. In vitro mutagenesis was used to alter a T to a C at position
9 of the tRNA[Ser]Sec gene or to alter an A to a G at the
nucleotide immediately 3' to the anticodon at position 37 using the
same 2.17-kb StuI-PvuII fragment, and these
fragments were used to develop additional colonies of transgenic
animals encoding either the "wild type" (i.e., the T-to-C
transition position at position 9) or the i6A-deficient (position 37)
transgene in FVB/N mice. Transgenic mice were derived by pronuclear
microinjection of fertilized eggs as previously described
(8). Tissues and organs were taken from sacrificed mice,
immediately placed into liquid nitrogen, and stored at
80°C until
ready for use.
Southern blot analysis and gene copy number.
Genomic DNA was
isolated from mouse tails (38) as modified by Promega,
digested with XhoI, electrophoresed on 1% agarose gels, and
transferred to a nylon membrane, and the membrane was cross-linked in
an UV-Stratalinker (from Stratagene) by standard techniques. The
membrane was hybridized with a 32P-labeled 240-bp fragment
encoding the Bluescript II vector DNA that was integrated into the
mouse genome as part of the transgene (see Fig. 1), and after obtaining
an autoradiogram, the filter was stripped and then hybridized with a
32P-labeled 193-bp fragment of human DNA encoding the
tRNA[Ser]Sec gene (43). Probes were labeled
with [
-32P]dCTP using a random primer labeling kit
(Stratagene) and used in hybridization assays, the resulting membranes
were washed, and autoradiograms were prepared as described previously
(40). This procedure was used to establish transgene
number in mice encoding a low copy number (2 to 4 transgenes).
Isolation and aminoacylation of tRNA, RPC-5 chromatography, and Northern blot analysis. Total tRNA was isolated from tissues, prepared for aminoacylation, and aminoacylated with [3H]serine under limiting tRNA conditions (21), and the resulting labeled seryl-tRNA was chromatographed twice on an RPC-5 column (29), first in buffer without Mg2+ and then in buffer with Mg2+ (9, 15, 22, 40). Seryl-tRNASer is more hydrophobic than seryl-tRNA[Ser]Sec in the absence of Mg2+ and therefore elutes later on the RPC-5 column, and it is less hydrophobic in the presence of Mg2+ and therefore elutes earlier. Thus, the tRNASer and tRNA[Ser]Sec populations can be chromatographically resolved from each other and quantitated following labeling with [3H]serine as described previously (9, 15, 22, 40).
Northern blot analysis of GPX1, GPX4, D1, TR1, SPS2, and SelP mRNAs was carried out by isolating total RNA from liver and kidney using an RNeasy minikit (according to the vendor's instructions). The RNA was electrophoresed on a 1% formaldehyde-agarose gel and transblotted to a nylon membrane. Filters were probed with a 32P-labeled bovine GPX1 cDNA EcoRI-HindIII fragment (28), and several IMAGE Consortium (LLNL) cDNA clones were generated as a MluI-SalI fragment encoding the SPS2 gene (IMAGE Consortium Clone ID 791719 [accession number {AN} AA414662]) and as NotI-EcoRI fragments encoding the D1, TR1, SelP, and GPX4 genes (IMAGE Consortium Clone ID 677180 [AN AA212899], 676579 [AN AA209061], 777018 [AN AA276440], and 1364475 [AN AI006169]), respectively. Membranes were stripped and reprobed with 32P-labeled human
-actin cDNA probe.
Primer extension.
The identity of the nucleotide at
position 9 in the tRNA[Ser]Sec transgene was
used to distinguish the contributions of the product from the
transgenes and that of the wild-type genes to the total tRNA[Ser]Sec population in transgenic mice by primer
extension. An oligonucleotide, 5'-GCCTGCACCCCAGACCACTGA-3',
that was complementary to bases 12 through 32 within the
tRNA[Ser]Sec gene was 5'-end labeled with
[
-32P]ATP and polynucleotide kinase, the unlabeled
nucleotide was removed with a NACS PREPAC column, and the resulting
labeled oligonucleotide was used as a primer in primer extension
studies as described previously (40). The extension buffer
included ddATP, resulting in termination at the first U in the tRNA
template. Relative intensities of bands were determined using a Bio-Rad
GS-710 calibrated imaging spectrophotometer.
Labeling of selenoproteins.
Transgenic and wild-type mice
were injected intraperitoneally with 50 µCi of 75Se/g of
body weight and sacrificed at 48 h after injection, and tissues
and organs were excised and immediately placed into liquid nitrogen and
stored at
80°C until ready for use. Tissues were homogenized in a
solution containing 40 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 0.5 mM
4-(-2-aminoethyl)benzenesulfonyl fluoride, sonicated for 2 min, and
centrifuged at 4°C for 20 min. Supernatants were electrophoresed on
sodium dodecyl sulfate-polyacrylamide gels, separated proteins were
transferred to nylon membranes, and transblots were exposed to a
PhosphorImager as described previously (20, 40). Gels were
stained with Coomassie blue.
Selenoprotein assays. GPX1 (40) and GPX3 (32) activities were assayed as previously described and measured as the nanomoles of NADPH oxidized/minute/milligram of protein using H2O2 as substrate. 5'-Deiodinase activity was measured using 125I-reverse T3 or 125I-T4 for type 1 (D1) or type 2 (D2) deiodinase, respectively, as previously described (3, 18). Thioredoxin reductase activity was determined in the presence of Escherichia coli thioredoxin using the insulin reduction method (1) in tissue extracts prepared as described below.
SelP, TR1, TR3, GPX4, and SelT were all measured by Western analysis. In addition, TR1 and TR3 were measured by 75Se labeling. SelP was measured using antibody #695 as described previously (24). For thioredoxin reductase assays, 0.8 g of 75Se-labeled mouse liver from each type of transgenic line were sonicated in 5 volumes of 25 mM Tris-HCl (pH 7.5)-1 mM EDTA-1 mM phenylmethylsulfonyl fluoride-5-µg/ml aprotinin-5-µg/ml leupeptin-5-µg/ml pepstatin A. After centrifugation, the supernatants were separately applied onto 0.5 ml ADP-Sepharose columns. The columns were washed with 0.5 ml of 25 mM Tris-HCl buffer (pH 7.5)-1 mM EDTA-0.15 M NaCl, and the proteins were eluted with 1 ml of 25 mM Tris-HCl (pH 7.5)-1 mM EDTA-1.0 M NaCl. The eluted fractions were tested for thioredoxin reductase activity and also analyzed by immunoblot assays with rabbit polyclonal antibodies specific for TR1 and TR3 (45) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses followed by PhosphorImager assays to determine the 75Se content (20). All thioredoxin reductase analyses were performed in parallel for the entire set of samples. SelT and GPx4 were analyzed with rabbit polyclonal antibodies raised against the C-terminal peptide of SelT (31) or an internal peptide of GPX4 (antibodies were kindly provided by Donna Driscoll), respectively. Crude extracts used for these assays were prepared as for the thioredoxin reductase analyses. X-ray films were quantified with a densitometer.Blood and selenium analyses. Blood samples were taken from mice by venal eye puncture. The serum was obtained by centrifugation and used for determining cholesterol, triglycerides, liver function (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and total protein) and kidney function (creatinine, urea nitrogen, and uric acid) in the National Institutes of Health Clinical Center using standard techniques. The amount of selenium in plasma or soft tissues was determined by automated electrothermal atomic absorption spectrometry using a Varian Spectra AA600 (Varian Instruments, Inc., Walnut Creek, Calif.) equipped with Zeeman-effect background corrections. Soft tissues were homogenized in 10% HNO3, allowed to digest for 48 h at room temperature, and centrifuged (1,000 × g), and selenium was analyzed in the supernatant. Samples of plasma or tissue digests were mixed with 3 volumes of a matrix modifier solution (1.25% Ni[NO3]2.6H2O, 0.09% PdCl2, 0.1% Triton X-100). Absorption was measured at 196.3 nm with a 2.0-nm slit signal; peak area is calibrated automatically using aqueous solutions of Na2SeO3 as standards. The limit of detection of this method is ca. 5 pg of Se, which yields a practical detection limit of approximately 20 ng of selenium/ml of sample. Quality control was effected using multiple aliquots of exhaustively analyzed human plasma as external control samples with a coefficient of variation of >7% (for duplicate analyses) used as the criterion for acceptance of all sample results. That criterion was derived experimentally using the variance components analysis described previously (37).
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RESULTS |
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Transgenic animals.
Transgenic mice were independently
generated using two tRNA[Ser]Sec gene constructs that
differ from each other by a single pyrimidine transition (U
C) at
position 9. C at position 9, which corresponds to wild-type chicken and
Xenopus tRNA[Ser]Sec (33),
permitted us to assess the levels of tRNAs derived from transgenes
relative to the endogenous tRNA[Ser]Sec population.
Transgenic mice were also generated by the introduction of a third
tRNA[Ser]Sec gene containing a purine transition (A
G)
immediately 3' to the anticodon at position 37 (see reference
26 and references therein for numbering of
tRNA[Ser]Sec nucleotide positions). The change at this
position prevents the formation of the highly modified base,
i6A (30), that normally occurs at this site.
The cloverleaf model of tRNA[Ser]Sec, as well as the two
described altered sites, are shown in Fig. 1A.
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C change at position 9. They were designated
+/+/TGWT2 and +/+/TGWT2/TGWT2 and +/+TG"WT"10 and
+/+/TG"WT"10/TG"WT"10, respectively (see Table 1). Three
founders were obtained with the alteration at position 37 (i6A
), and the resulting mouse lines
generated from these founders contained 2 to 4, 8 to 16, and 20 to 40 transgenes, respectively, and were designated
+/+/TGi6A
2 and
+/+/TGi6A
2/TGi6A
2,
+/+/TGi6A
8 and
+/+/TGi6A
8/TGi6A
8,
and +/+/TGi6A
20 and
+/+/TGi6A
20/TGi6A
20
(Table 1).
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Analysis of the tRNA[Ser]Sec population.
To
assess changes in the tRNA[Ser]Sec population in animals
bearing the above-described transgenes, tRNA was prepared from the
liver, kidney, brain, and testes of transgenic wild-type and sibling mice. Isolated tRNA was aminoacylated with [3H]serine,
resulting in the labeling of tRNASer and the
mcm5U and mcm5Um tRNA[Ser]Sec
isoforms. The amounts of the Sec isoacceptors relative to the seryl-tRNA population were determined by RPC-5 chromatography, where
the unmethylated isoform, mcm5U, elutes first and the
methylated form, mcm5Um, elutes second from the column (see
Materials and Methods) (9, 15, 22, 40). A typical
chromatographic separation of the Sec isoforms from livers of +/+,
+/+/TGWT2, and +/+/TGWT2/TGWT2 sibling mice is shown in Fig.
2. The tRNA[Ser]Sec
population increased with increasing wild-type gene copy numbers in the
livers of transgenic animals, and the relative distributions of
mcm5U and mcm5Um were altered, albeit slightly.
The relative amounts of the seryl-tRNA[Ser]Sec population
and the distributions of the mcm5U and mcm5Um
isoacceptors from each selected organ were determined in this manner.
The data from liver, kidney, brain, and testes of transgenic mice
harboring wild-type tRNA[Ser]Sec transgenes are
summarized in Table 2, and those from
transgenic mice harboring the i6A
transgenes
are summarized in Table 3.
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Overexpression of the wild-type tRNA[Ser]Sec population. The increase in the tRNA[Ser]Sec population was clearly not directly proportional to gene copy number in transgenic animals carrying wild-type transgenes, nor was it the same in all tissues. For example, the increase was about 3.5-fold in the liver but more than 6-fold in brains of mice carrying 20 extra tRNA[Ser]Sec copies (Table 2). In addition, the relative distributions of the two tRNA[Ser]Sec isoforms in tissues of mice carrying wild-type transgenes were altered as gene copy numbers increased. As shown in Table 2, the amount of mcm5U relative to that of mcm5Um increased slightly in the TGWT2 animals, but this effect was more dramatic in the TG"WT"10 animals. These observations support the hypothesis that the methylase responsible for converting mcm5U to mcm5Um is likely to be limiting (see also references 23 and 40).
The introduced nucleotide change at position 9 in the tRNA[Ser]Sec transgene permitted us to distinguish the amount of gene product contributed to the tRNA[Ser]Sec population by the transgenes relative to that from the host genes by primer extension. Total tRNA from livers, kidneys, and testes of transgenic mice and their wild-type siblings was used as a template to extend the sequence of an oligonucleotide complementary to positions 12 through 32. Since ddATP replaced dATP in the extension buffer, primers were extended until a U was encountered in the template tRNA. The primer was therefore extended only three nucleotides when wild-type tRNA[Ser]Sec was used as a template and six nucleotides when transgene-derived tRNA[Ser]Sec was used as a template. As expected, the oligonucleotide extended only three nucleotides in tRNA samples isolated from livers, kidneys (Fig. 3A, lanes 2 and 5, respectively), and testes (data not shown) of the wild-type siblings. In contrast, total tRNA from these same tissues obtained from heterozygous and homozygous transgenic animals contained the expected extension products for tRNA transcribed from the transgenes (Fig. 3A, lanes 3, 4, 6, and 7, respectively).
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Expression of the i6A
transgenes.
The tRNA[Ser]Sec population was examined in selected
organs of transgenic mice containing tRNA[Ser]Sec genes
engineered to be incapable of forming the i6A modification.
The i6A
tRNA[Ser]Sec elutes
earlier from the RPC-5 column than the endogenous
i6A-containing tRNA[Ser]Sec population
(10, 30, 42, 47) while retaining its ability to be
aminoacylated (42, 47). These observations facilitated the
analysis of each of the tRNA[Ser]Sec species for the
liver, kidney, brain, and testes. The total amount of the host
tRNA[Ser]Sec population remained virtually unchanged in
livers, kidneys and brains of these mice, even though the
i6A
form increased to levels as high as about
30 to 40% of the total tRNASer population in mice carrying
the highest transgene copy number (Table 3). In testes, the level of
endogenous tRNA declined slightly as the level of
i6A
tRNA increased. Examination of the
distributions of mcm5U and mcm5Um in animals
expressing i6A
tRNA[Ser]Sec
indicated that there was an increase in mcm5U with a
proportional decline in mcm5Um in the livers, kidneys, and
brains of these animals. The data also suggest that the levels of
wild-type tRNA[Ser]Sec and i6A
tRNA[Ser]Sec are regulated independently of each other
and that the level of the tRNA[Ser]Sec population is
determined in these tissues, at least in part, by a feedback mechanism
governed by the isoforms containing i6A at position 37 (see Discussion).
Protein synthesis. Selenoprotein biosynthesis was assessed in the transgenic mice described above by injection with 75Se. Proteins from livers, kidneys, testes, brains, muscles, and hearts of these and control animals were isolated following labeling with 75Se and examined by gel electrophoresis. Coomassie blue-stained gels of total proteins from these tissues showed only minor differences in protein patterns in transgenic mice containing 10 to 20 wild-type transgenes compared to results for their wild-type, nontransgenic siblings (data not shown). PhosphorImaging, used specifically to detect 75Se-labeled selenoproteins, also failed to detect significant differences between transgenic and corresponding control tissues. Therefore, the higher levels of tRNA[Ser]Sec resulting from the expression of 2 to 4 and 10 to 20 wild-type transgene copies had little or no effect on either general protein synthesis or selenoprotein levels (data not shown).
In contrast to the data presented above, the presence of i6A
tRNA[Ser]Sec caused
considerable changes in selenoprotein synthesis. Seven tissues,
including the cerebellum, were excised from 75Se-labeled
mice, and the resulting protein extracts were electrophoresed. Coomassie blue staining of total protein within gels showed some variations in tissue extracts from either wild-type or heterozygous and
homozygous i6A
tRNA[Ser]Sec
transgenic mice (Fig. 4A, C, and E). No
consistent differences were observed, however, when
duplicate samples of sibling mice were analyzed (data not shown). In
contrast, 75Se-labeled proteins were significantly altered
in the tissues of these mice, with selenoprotein patterns being
different in each of the seven tissues examined (Fig. 4B, D, and F).
For example, there was an apparent decrease in GPX1 in the livers
of transgenic mice carrying two or more
i6A
tRNA[Ser]Sec
transgenes (Fig. 4B, D and F) and in kidneys of mice carrying eight or more i6A
tRNA[Ser]Sec
transgenes (Fig. 4D and F). GPX1 levels appeared to be less affected in
kidney than liver in mice with genotype
+/+/TGi6A
8 or
+/+/TGi6A
8/TGi6A
8
(Fig. 4D).
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tRNA[Ser]Sec exhibited more GPX1 activity than those
expressing more of the i6A
tRNA[Ser]Sec (Table 4). A similar dose-dependent effect
was observed for the inhibition of GPX3, which is synthesized in the
kidney and secreted into plasma. The effects of the expression of
i6A
tRNA[Ser]Sec on GPX4,
deiodinase 1 (D1 synthesized in the liver), deiodinase 2 (D2,
synthesized in the pituitary gland), and SelP (synthesized primarily in
the liver and secreted to plasma), TR1, and SelT (31) are
also presented in Table 4. The tissue specificity of these effects is
particularly apparent in the testes, where as much as an 80% reduction
in GPX1 was observed while the levels of GPX4, TR3, and SelT were
either unaffected or stimulated.
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Northern blot analysis.
The data presented above cataloging
the reduced amounts of selenoproteins in organs of transgenic mice
could be explained by effects on either transcription or translation.
Therefore, we examined the mRNA levels of several selenoproteins by
Northern analysis. GPX1 activity was dramatically reduced in
i6A
tRNA[Ser]Sec-expressing
animals, as judged by both 75Se labeling and direct enzyme
assay. GPX1 mRNA levels were measured by Northern blot analysis in
liver and kidney tissue of transgenic mice, where it was apparent that
the amount of GPX1 mRNA was virtually unchanged in these two tissues,
with the possible exception in livers of mice carrying the highest
number of mutant tRNA[Ser]Sec transgenes (Fig.
5A). We also examined several other
selenoprotein mRNAs, which included SelP, TR1, D1, SPS2, and
GPX4 from livers of mice expressing i6A
tRNA
transgenes (Fig. 5B). In general, any differences observed in
selenoprotein mRNA levels in organs from i6A
mice were insufficient to account for the reduction observed in the
corresponding selenoproteins. The data therefore indicate that the
defect in selenoprotein biosynthesis caused by expression of the
i6A-deficient tRNA[Ser]Sec occurs
at the translation step. Warner et al. (47) have also shown that CHO cells transfected with an i6A
tRNA[Ser]Sec gene exhibited reduced type 1 deiodinase
levels without affecting steady-state levels of the corresponding mRNA.
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Selenium levels and blood chemistries.
Selenium levels in
livers, kidneys, brains, testes, and serum of selected transgenic mice
and their wild-type siblings were analyzed (Table
5). Dramatic differences in selenium
levels between the i6A
mice and their sibling
controls were observed in plasma (30 to 60%), liver, kidney, and
heart, while the difference was less apparent in the testes and brain.
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transgenes as
evidenced by their phenotypic appearance or behavior. Their blood
chemistries (see Materials and Methods) fell within the normal ranges
of their wild-type siblings regarding cholesterol, serum triglycerides,
and liver and kidney function tests (data not shown).
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DISCUSSION |
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There is a growing appreciation of the diverse biological roles for selenoproteins and the central role for tRNA[Ser]Sec in their synthesis. To study the regulation of selenoprotein biosynthesis, we generated transgenic mice that expressed elevated levels of either wild-type or i6A-deficient tRNA[Ser]Sec and examined the consequences of these manipulations on selenoprotein and tRNA[Ser]Sec levels. The generation of the animals described in this study provides the first example of the production of transgenic mice in which multiple copies of a tRNA gene are stably introduced and expressed.
It is evident from our analyses of the tRNA[Ser]Sec
population in several tissues from these transgenic mice that they can
tolerate relatively high levels of tRNA[Ser]Sec, since
some tissues exhibited increases of more than 6-fold in wild-type
tRNA[Ser]Sec or more than 12-fold in
i6A
tRNA[Ser]Sec without
apparent ill effects on their health. The use of primer extension
permitted the independent examination of endogenous tRNA[Ser]Sec and that derived from the wild-type
transgenes, and the data indicated that both contribute to the
resulting tRNA population. Using this approach, we observed a reduction
in endogenous tRNA[Ser]Sec with increasing amounts of
tRNA[Ser]Sec derived from wild-type transgenes (Fig. 3).
Levels of i6A-deficient tRNA[Ser]Sec could be
directly evaluated by RPC-5 chromatography, and quantities were
correlated with gene copy number. Levels of i6A-deficient
tRNA[Ser]Sec as high as 40% of the total
tRNASer population did not affect the level of
tRNA[Ser]Sec expressed from the corresponding endogenous
genes. The distributions of mcm5U and mcm5Um,
however, were dramatically affected with increasing amounts of
i6A
tRNA[Ser]Sec. Their
distributions mimicked those seen in liver and kidney tissue of
selenium-deficient rats and mice and in mammalian cells grown in
selenium-deficient medium, where mcm5Um was significantly
reduced (9, 15, 22). Collectively, these observations
support the existence of a feedback mechanism of control that limits
the amounts of tRNA[Ser]Sec found in particular tissues.
The effector(s) involved in this feedback control likely requires the
i6A modification at position 37. The data also show that
the presence of the i6A
tRNA[Ser]Sec results in an inhibition of the maturation process.
For mice overexpressing wild-type tRNA[Ser]Sec, the increase was largely restricted to the mcm5U isoform in the tissues examined. There was no apparent effect on selenoprotein synthesis. These data are consistent with the methylation of tRNA[Ser]Sec on the ribosyl moiety at position 34 being a limiting step in tRNA maturation (40). As noted previously in both cell culture (10, 22) and animal models (9, 15), the distribution of the two major isoforms of mammalian tRNA[Ser]Sec responds to increased selenium availability with a characteristic increase in mcm5Um and translational induction of GPX1. This, and the restrictions observed in the conversion to the mcm5Um seen in vivo, suggest that the balance between tRNA[Ser]Sec isoacceptors serves a biologically significant purpose that is yet to be defined.
In contrast to the data obtained with the overexpression of wild-type
tRNA[Ser]Sec, selenoprotein synthesis was dramatically
affected in mice expressing i6A
tRNA[Ser]Sec. This was demonstrated by 75Se
labeling of selenoproteins and by direct enzyme assay and/or Western
blot analyses. The expression of several selenoproteins was
reduced substantially in all tissues examined (e.g., GPX1), with a
dose-dependent effect being evident. All selenoproteins examined
in this study appeared to be reduced in the livers of i6A
mice, with the exception of TR3, which
was either unaffected or stimulated. The expression of other
selenoproteins, such as GPX4 and SelT, was also inhibited in the liver
but unaffected in the testes by increasing amounts of
i6A
tRNA[Ser]Sec. The lack of
an effect in the testes may be due to the fact that the
tRNA[Ser]Sec population in the testes (>7% of the total
serine tRNA population) is substantially higher than that in the liver
(~2.5% of the total serine tRNA population). Therefore, the
i6A-deficient tRNA[Ser]Sec population
represented a much lower proportion of the tRNA[Ser]Sec
population in the testes than in the liver (Table 3). Translation of
GPX1 appeared to be particularly sensitive to the presence of
i6A
tRNA[Ser]Sec, consistent
with the possibility that its synthesis may be more dependent on the
mcm5Um isoform than is that of other selenoproteins
(9).
A hierarchy exists with regard to the effects of selenium deficiency on
the maintenance of individual selenoproteins as well as selenium
retention by different organs (2, 24, 34, 36, 39). For
example, GPX1 activity was reduced to 1% in liver tissue and about 4 to 9% in kidney, heart and lung tissues during selenium deficiency in
rats. GPX4 activity, on the other hand, was reduced only about 25 to
50% in these tissues but was unaffected in the testes. A similar
hierarchy of selenium maintenance may be occurring in response to the
expression of i6A
tRNA[Ser]Sec.
As seen in Table 4, the levels of each of the selenoproteins analyzed
in i6A
mice were reduced to different
degrees, and the extent of the reduction differed depending on the
organ examined.
The greater sensitivity of GPX1 activity to selenium deficiency was
attributed in large part to increased mRNA turnover (11, 34,
44). In contrast, the reduction in GPX1 observed with increasing
amounts of i6A-deficient tRNA is not likely due to mRNA
turnover, since GPX1 mRNA stability was not significantly altered in
kidneys of i6A
mice. The stem-loop structure
in the 3' untranslated region of mammalian selenoprotein mRNAs,
designated the SECIS element (35), has also been shown to
play a role in establishing a selenoprotein hierarchy
(36). Thus, it would appear that there are several levels
of regulation involved in determining the priority of selenoprotein synthesis under various biological conditions.
Under conditions of selenium deprivation in the diets of rats and mice,
the levels of this element are reduced in the liver and kidneys, while
the brain and testes retain most of their selenium (2,
25). Transfer RNA[Ser]Sec maturation and
selenoprotein synthesis are responsive to selenium status, and thus
these two parameters are more affected by change in selenium status in
the liver and kidneys than in the testes and brain (15,
25). Selenoprotein biosynthesis was most affected in the liver,
kidneys, and brain in the presence of i6A
tRNA[Ser]Sec and least affected in the testes, while
selenium losses were highest in the liver and kidneys and lowest in the
testes and brain. The losses in selenium in the brain were the lowest
of the tissues examined, and this reduction may reflect only an
inhibition in selenoprotein biosynthesis. Observations showing a
differential loss in selenium retention and differential rates in
selenoprotein biosynthesis suggest that the
i6A
tRNA[Ser]Sec transgenic
mice can be used as a model system to better understand the hierarchy
in selenium retention by different tissues.
There is an abundance of literature supporting selenium as a protective
agent against a variety of mutagens, carcinogens, and viruses in
laboratory animals, namely rats and mice (reviewed in reference
19). Animals given slightly elevated levels of selenium in
their diets have the greatest protection upon exposure to these
environmental stresses. Studies with humans also support a role of
selenium as a beneficial micronutrient in the diet (17). It is unclear whether these beneficial effects of selenium on health
are due to selenium-containing, low-molecular-weight compounds (see
references 17 and 27 and references therein) or to
selenium-containing proteins (reviewed in reference 19).
Since transgenic i6A
tRNA[Ser]Sec mice have reduced selenoprotein levels, it
will be of considerable interest to determine if slightly elevated
levels of dietary selenium will afford these transgenic mice the same
protection from environmental stress as their wild-type siblings have.
Since the i6A
tRNA[Ser]Sec mice
selectively express selenoproteins, then by controlling the mutant
tRNA[Ser]Sec gene dosage, these animals may be used to
provide a useful model for resolving the roles of individual
selenoproteins as well as their overall influence on health. It is of
interest that adult animals made selenium deficient through diet appear
to be as healthy as their control counterparts maintained on
selenium-sufficient diets unless they are challenged or stressed
environmentally. The i6A
tRNA[Ser]Sec transgenic mice also appear to be as healthy
as their wild-type littermates. By the selective use of individual
carcinogens, mutagens, and viruses and careful monitoring of the rates
of malignant change at target sites, these transgenic mice may be used
as a powerful tool for determining the role of individual
selenoproteins in protecting against specific environmental stresses.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported in part by NIH grants GM616603 (V.N.G.), CAA81153 (A.M.D.), DK47320 (M.J.B.) and ES02497 (R.F.B. and K.E.H.) and a grant 99A026 from the American Institute for Cancer Research (A.M.D.) and from the Molecular Medicine Research Group Program, Ministry of Science and Technology of Korea (B.J.L.).
The authors express their sincere appreciation to Glen Merlino for his advice and helpful discussions throughout the course of this study.
M.E.M. and B.A.C. contributed equally to this project.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: National Cancer Institute, National Institutes of Health, Building 37, Room 2D09, Bethesda, MD 20892. Phone: (301) 496-2797. Fax: (301) 435-4957. E-mail: hatfield{at}dc37a.nci.nih.gov.
| |
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