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Department of Human Nutrition, University of Illinois at Chicago, Chicago, IL 60612;
*
Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Institutes of Health, Bethesda, MD 20892; and
Department of Kinesiology, University of Illinois at Chicago, Chicago, IL 60612
3To whom correspondence should be addressed. E-mail: adiamond{at}uic.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • selenium • tRNA • selenoprotein • selenocysteine • translation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Selenium is cotranslationally incorporated into selenoproteins as the amino acid selenocysteine in response to certain in-frame UGA codons in selenoprotein mRNAs [reviewed in (2
)]. In eukaryotes, translation of UGA as selenocysteine is dependent on the presence of a selenocysteine insertion sequence (SECIS)4
element in the 3'-untranslated region of selenoprotein mRNAs, as well as a number of dedicated translation factors, including the selenocysteine elongation factor, the SECIS element binding protein-2 (SBP2) and selenocysteine tRNA (3
–6
). This tRNA is initially aminoacylated with serine by seryl-tRNA synthetase, and the serine is converted into selenocysteine while attached to the tRNA; hence, it is designated tRNA[Ser]Sec (7
). At 90 nucleotides, tRNA[Ser]Sec is the longest eukaryotic tRNA sequenced to date, and contains only four modified nucleotides. Two major tRNA[Ser]Sec isoforms have been characterized in mammalian cells; these differ by 2'-O-methylation of the ribose portion of methylcarboxymethyl-5'-uridine (mcm5U) in the wobble position of the anticodon, resulting in methylcarboxymethyl-5'-uridine-2'-O-methylribose (mcm5Um) (8
). This methylation causes a dramatic change in the conformation of tRNA[Ser]Sec from a relative compact structure to a more open one (8
).
The relative abundance of the two tRNA[Ser]Sec isoforms varies among different tissues and cell types. For example, rat testes have been shown to contain predominantly the unmethylated isoform, whereas the mcm5Um-containing isoform predominates in the brain (8
). In addition, the distribution between these two tRNA[Ser]Sec species often reflects selenium availability. Typically, selenium supplementation results in an increase in both the total tRNA[Ser]Sec population and the relative abundance of the methylated isoform (9
). Selenium has also been shown to increase the stability of tRNA[Ser]Sec when Xenopus oocytes were injected with either the Xenopus tRNA[Ser]Sec gene or in vitro synthesized tRNA[Ser]Sec (10
).
The abundance of the methylated and unmethylated tRNA[Ser]Sec appears to be tightly regulated in mammalian cells (11
). Overexpression of tRNA[Ser]Sec in Chinese hamster ovary (CHO) cells after transfection of multiple copies of the corresponding gene resulted in a proportional increase in the total amount of tRNA[Ser]Sec. However, the majority of the expanded tRNA[Ser]Sec population remained in the mcm5U-containing form even though the tRNA transcribed from the transfected gene was capable of serving as a substrate for methylation (11
). Similar results were observed in transgenic mice (12
). Conversely, reduction in the tRNA[Ser]Sec gene copy number by homologous recombination in embryonic stem cells resulted in a proportional decline in the total tRNA[Ser]Sec population, predominantly through decrease of the mcm5U-containing isoform (5
,13
). In each of these approaches, the level of the mcm5Um-containing isoform tended to be maintained despite changes in the amounts of tRNA[Ser]Sec population achieved by genetic manipulation.
The regulation of the distribution of tRNA[Ser]Sec isoforms is not understood. Different mechanisms of regulation, including transcription, methylation and RNA stability may be involved. To explore this issue, the de novo transcription of tRNA[Ser]Sec in CHO cells was inhibited and the levels of each isoform monitored. These studies indicated that the turnover of tRNA[Ser]Sec is rapid relative to total tRNA, and that it is attenuated by the addition of selenium. These studies suggest that tRNA turnover may be an important factor in the regulation of selenoprotein biosynthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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CHO cells were grown in modified Eagle’s medium-
(MEM; Invitrogen, Carlsbad, CA) with 100 mL/L fetal bovine serum (FBS) (Bio-Whittaker, Walkersville, MD) and 10 mg/L penicillin/streptomycin (Invitrogen) in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. For all of the experiments presented, independent cultures were either untreated, treated with 10 mg/L actinomycin D (Act D; Sigma, St. Louis, MO) in 0.5% ethanol, and/or 30 nmol/L sodium selenite (Sigma) as indicated in the text. Exposure time to Act D did not exceed a total of 10 h because floating cells indicative of toxicity became apparent at longer exposure times.
Northern blot analysis of tRNA.
Total tRNA was extracted from CHO cells as described (14
), isolated using DEAE cellulose and quantified by optical density at 260 nm. tRNA (5 µg/sample) was electrophoresed on 10% polyacrylamide/7mol/L urea gel (acrylamide/bis-acrylamide, 30:1) and transferred by capillary action to BioTrace Nitrocellulose (0.45 µm) (Pall Life Sciences, Ann Arbor, MI) or Gene Screen Plus membranes (Perkin Elmer, Boston, MA) according to the product guidelines. Filters were hybridized with 32P-labeled probes representing either tRNA[Ser]Sec or serine transfer RNA (tRNASer). A probe for tRNA[Ser]Sec was generated by random primed labeling (15
) from a 193-nucleotide template containing the human tRNA[Ser]Sec gene (16
). Double stranded complementary tRNASer (GENBANK accession number M38616) was generated by annealing two complementary oligonucleotides, one of which corresponded to 24 nucleotides of the 5' end of tRNASer with three additional nonbase-pairing guanosines at the 5' end: (5'-GGGCAGGTTCGAATC CTGCCGACTACG-3') and a second oligonucleotide, complementary to the first 24 nucleotides of the first primer (5'-CGTAGTCGGCAGGATTCGAACCTG-3'). A probe was generated by annealing the oligonucleotides and extending the 3' end of the second oligonucleotide with Klenow fragment (Boehringer Mannheim, Basel, Switzerland), 2.96 x 106 Bq 32P-dCTP (Perkin Elmer, 3000 Ci/mmol), and 200 µmol/L each dATP, dGTP and dTTP (Invitrogen). Hybridization was performed as described by the manufacturers’ recommendations. Washing conditions for the tRNA[Ser]Sec hybridization were 0.1 X SSC, 1g/L SDS at 65°C for 3 h (Gene Screen Plus membrane) and 10 g/L bovine serum albumin, 50 mmol/L NaPO4 (pH 7.0) and 70 g/L SDS for three 20-min washes at room temperature followed by three 20-min washes at 65°C in 1.0 mol/L Tris, pH 8.0, 1.5mol/L NaCl (BioTrace Nitrocellulose). Wash conditions for the tRNASer hybridization on Gene Screen Plus were two 1-h washes in 2 X SSC, 1 g/L SDS at 55°C and the same as used for the tRNA[Ser]Sec probe on BioTrace Nitrocellulose, but with the last three washes at 55°C. Bands visualized in autoradiograms were quantified by densitometry using a Bio Rad 710 laser densitometer (Bio Rad Laboratories, Hercules, CA).
Transfection of CHO cells with the mouse tRNA[Ser]Sec gene.
To engineer cells that overproduce tRNA[Ser]Sec, CHO cells were transfected with a mouse cDNA for the tRNA essentially as described (11
). A 1.93-kbp Xho I-Stu I fragment excised from pBluscript II KS (17
) was cotransfected with the pLNCX vector (18
) into CHO cells with Lipofectin (Invitrogen), as indicated by the manufacturer, using 10 µL of Lipofectin and a total of 2.25 µg of DNA per 5 x 105 cells. Cells at 90% confluence were washed with Opti-MEM serum-free medium (Invitrogen), overlaid with the Lipofectin-DNA-Opti-MEM mixture in a volume of 2.0 mL, and incubated for 6 h. The cells were then incubated for 24 h in standard medium, trypsinized and replated at 1:50 or 1:100 in 500 mg/L G418 (Sigma) for 10 d, at which time G418-resistant colonies were isolated and expanded to mass culture for analysis.
Primer extension analysis of tRNA[Ser]Sec.
The relative abundance of tRNA[Ser]Sec transcribed from the host hamster gene, and that produced from the transfected mouse gene, were determined by a primer extension assay as described (11
). This assay exploits nucleotide differences between the two genes at positions 13 and 14, where the CHO gene is 11UCUCC (15
) and the mouse gene is 11UCCUC (15
). Briefly, a 5' end-labeled primer complementary to the 5' end of both the hamster and mouse genes was annealed to 100 ng of total tRNA and extended with Superscript 2 Reverse Transcriptase (Invitrogen) in the presence of dATP, dGTP, dCTP and dTTP (Invitrogen). The extension of the mouse sequence terminated at position 14, whereas the endogenous hamster gene yielded an extension product one nucleotide longer. After the reaction, equal amounts of radioactivity were applied to a 15% polyacrylamide gel, and extension products were visualized by autoradiography. Bands were quantified by laser densitometry of autoradiograms obtained by the exposure of gels to X-ray film for periods of time needed to obtain visible, but not overexposed signals.
Chromatography of tRNA[Ser]Sec.
Total tRNA was isolated from 
1 g of CHO cells, deacylated, aminoacylated with [3H]serine, which labels both serine and selenocysteine tRNAs, and chromatographed on an RPC-5 column (19
) as described previously (11
). The aminoacylated tRNA was chromatographed twice on the RPC-5 column, first in the presence of Mg2+ and then in the absence of Mg2+, as described (20
). [3H]Seryl-tRNASer is less hydrophobic than [3H]-seryl-tRNA[Ser]Sec in the presence of Mg2+ and thus elutes earlier from the RPC-5 column; it is more hydrophobic in the absence of Mg2+ and thus elutes later from the column. These two chromatographic steps resolve seryl-tRNA[Ser]Sec from seryl-tRNASer and allow the quantification of the tRNA[Ser]Sec population relative to the total seryl-tRNA population as well as the determination of the distribution of the two tRNA[Ser]Sec isoforms.
Determination of glutathione peroxidase (GPx) activity.
GPx activity was measured using the standard coupled spectrophotometric method as previously described (21
). Briefly, cells were scraped and washed with ice-cold PBS and resuspended in ice-cold sodium phosphate buffer (0.1 mol/L, pH 7.0). Cells were lysed by sonication and debris was removed by centrifugation for 3 min at 11,000 x g. The assay contained 0.25 mmol/L NADPH, 5.0 mmol/L glutathione, 5.0 mmol/L EDTA, 1.0 U glutathione reductase, 5.0 mmol/L sodium azide, 5–100 µL lysate and 65 µmol/L hydrogen peroxide in a 1-mL reaction volume (Sigma, all reagents). The rate of oxidation of NADPH, corresponding to the change in absorbance at 339 nm, was measured in a Beckman DU 640B spectrophotometer (Beckman Coulter, Fullerton, CA) at 30-s intervals for 5 min. The results were adjusted by subtraction of the rate of oxidation in the absence of cell extract, and normalized to the amount of protein used in the assay. The results were expressed as nmol NADPH oxidized/(min · mg protein).
Measurement of total protein synthesis.
Total protein synthesis was measured by the method of Vandenburgh and Kaufman (22
) with the modification that 100 mL/L trichloroacetic acid (TCA) was used to precipitate proteins. Briefly, 5 x 104 CHO cells were transferred to -MEM medium (Gibco) containing 100 mL/L heat inactivated FBS (Bio-Whittaker) and 3.7 x 107 Bq/L 14C-labeled phenylalanine (Amersham, Uppsala, Sweden, 1.85 x 109 Bq/L) for 1 h, at which time the cells were lysed, and the protein precipitated with TCA. Total protein concentration was measured by the Lowry method using the Bio Rad DC (Detergent Compatible) protein assay, according to the manufacturers instructions and radioactivity was quantified using a scintillation counter.
Statistical Procedures.
Where indicated in the text, means are derived from at least three independent experiments and error bars represent the standard deviation. The evaluation of statistical significance was tested by two-way ANOVA and Tukey’s Studentized Range (HSD) Test for y (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To determine the relative stability of tRNA[Ser]Sec compared with the total tRNA population, its abundance in CHO cells was evaluated by Northern blot analysis after the inhibition of de novo transcription with 10 mg/L Act D for 10 h (Fig. 1
). Because equal amounts of total tRNA from each sample were applied to the gels, the intensity of the bands represents the abundance of probed tRNA[Ser]Sec or tRNASer relative to the total tRNA population. Cells exposed to Act D for 10 h had 61% less tRNA[Ser]Sec, indicating that this tRNA had a rate of decay that was faster than the tRNA population as a whole. In comparison, no measurable decline in band intensity was observed when the same samples were analyzed using the tRNASer probe.
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Distribution of tRNA[Ser]Sec isoforms after exposure to ActD.
To assess changes in isoform distribution during transcriptional inhibition, total tRNA was extracted from either control cells, cells treated with Act D, selenium or both, and the relative distribution and abundance of the tRNA[Ser]Sec isoforms were determined by RPC-5 chromatography (Fig. 2
and quantified in Table 1
). In agreement with the Northern blot data presented above, RPC-5 chromatography indicated that selenium supplementation increased the total tRNA[Ser]Sec population from 1.64 to 2.45% of the total serine-labeled tRNA population. This was accompanied by an increase in the ratio of the methylated to unmethylated isoforms from 0.61 to 2.18. After a 10-h exposure to Act D, the relative abundance of the methylated isoform increased, with the ratio of the two forms increasing from 0.61 to 3.52. When the cells treated with Act D had been previously incubated in medium supplemented with selenium, the ratio of mcm5Um/mcm5U increased from 2.18 to 6.08. This shift in the ratios of the two forms was largely due to a sharp decline in the unmethylated isoform. When expressed as a percentage of the total serine-labeled tRNA, the methylated form of tRNA[Ser]Sec declined by only 5% after 10 h of Act D treatment in the control cells and 13% in the cells incubated in medium containing added selenium. In contrast, the portion of the serine-labeled tRNA represented by the unmethylated isoform declined by 83% in cells not incubated in supplemental selenium, and by 69% in cells incubated in selenium. These results indicated that a shift in the tRNA[Ser]Sec isoform distribution toward the methylated isoform accompanied transcriptional inhibition, that this was accentuated under conditions of added selenium, and was due mainly to a decline in the abundance of the unmethylated isoform.
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One means by which selenium could inhibit the decay of tRNA[Ser]Sec is through the existence of a specific degradative system that becomes saturated by tRNA[Ser]Sec in situations of increased selenium abundance. To test this possibility, stable CHO transfectants that overexpressed tRNA[Ser]Sec were generated by the transfection of a plasmid containing the gene for the mouse tRNA[Ser]Sec. The resulting increment of the levels of tRNA[Ser]Sec obtained by transfection was confirmed by Northern blotting, which indicated that these cells produced 10 times as much tRNA[Ser]Sec as control cells transfected with vector only (data not shown). These cells were incubated in either standard media or for 3 d in media supplemented with selenium, and exposed to Act D for 10 h.
The effect of increased gene copy number on the turnover of tRNA[Ser]Sec arising from the native gene and from the transfected gene was evaluated by primer extension. This analysis permitted the independent evaluation of tRNA[Ser]Sec derived from each gene source due to sequence differences between the two genes at positions 13 and 14 (Fig. 3A
). An extension product of 5 nucleotides (+5) resulted when the CHO tRNA[Ser]Sec was used as a template, whereas an extension product of 4 nucleotides (+4) was obtained when the tRNA[Ser]Sec was derived from the transfected mouse gene. Examination of the data in the figure and quantification of band intensities by densitometry (Fig. 3
B) confirm the data presented from both Northern analysis and RPC-5 chromatography, demonstrating that selenium inhibits the decline in the levels of tRNA[Ser]Sec abundance after transcriptional inhibition. In transfectants that overexpress tRNA[Ser]Sec, this effect of selenium was similar for tRNA[Ser]Sec transcribed from either the CHO or transfected mouse gene, and the rate of decline appeared to be faster with higher levels of the tRNA. To ensure that this result was not the consequence of primers becoming limiting in the primer-extension assay when tRNA[Ser]Sec was overexpressed, studies were performed indicating that tRNA[Ser]Sec does not become limiting, even when 10 times more tRNA template was used (Fig. 3
C). Collectively, these observations indicate that tRNA[Ser]Sec stability may be influenced by its abundance.
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A, lanes 6–9, +5 bands), and this observation is similar to that in transgenic mice containing multiple copies of the tRNA[Ser]Sec (12
). Effect of Act D on GPx induction and general protein synthesis.
It has recently been suggested that the different tRNA[Ser]Sec isoforms may contribute differently to selenoprotein translation (12
). This concept is supported by data indicating that increases in GPx activity observed after selenium supplementation are often accompanied by increases in mcm5Um levels (23
,24
). Because the chromatography data presented in Figure 2
and Table 1
indicated a shift in the relative abundance of the methylated tRNA[Ser]Sec isoform after Act D treatment, the effect of Act D exposure on the stimulation of GPx translation by selenium was examined. GPx levels have previously been shown to be stimulated four- to fivefold in CHO cells incubated for 3 d in selenium-supplemented medium (25
). Supplementation with 30 nmol/L sodium selenite for 10 h doubled GPx activity from the baseline level (7.78) to 15.85 nmol NADPH/(min · mg protein) (Fig. 4A
). Simultaneous incubation of cells for 10 h in medium containing supplemental selenium and Act D did not influence the stimulatory effect of selenium. These results might indicate that the distribution of tRNA[Ser]Sec isoforms is not relevant to GPx induction by selenium. However, this conclusion could be confounded by a general inhibition of protein synthesis by Act D in these cells. Therefore, the effect of Act D on the rate of total protein translation was assessed. Cells exposed to Act D for 10 h as described above were incubated in 14C-labeled phenylalanine for 1 h and the amount of TCA-precipitable radioactivity was determined (Fig. 4
B). Exposure to Act D had a general effect on protein synthesis, as previously observed (26
) and can be attributed to inhibition of mRNA binding to ribosomes (27
). Given this observation, it is not known whether the changes in tRNA[Ser]Sec levels and distribution in these cells are associated with the ability of selenium to stimulate GPx translation. Additional studies involving the genetic manipulation of tRNA[Ser]Sec isoacceptor levels are underway to address this issue.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The responsiveness of tRNA[Ser]Sec levels to selenium levels is therefore conserved across vertebrate evolution and is likely to be a mechanism by which the selenoprotein biosynthesis machinery can respond to varying selenium availability.
An additional tier of regulation in selenoprotein synthesis may derive from the maintenance of a portion of the total tRNA[Ser]Sec population in a form that contains mcm5Um in the wobble nucleoside position instead of mcm5U. As seen here and reported previously, supplemental selenium increased the relative abundance of the methylated isoform in addition to total tRNA[Ser]Sec. Here we report that the inhibition of total cellular transcription is accompanied by a dramatic shift in the ratio of the two isoforms toward the methylated form, and that this shift is accentuated in cells supplemented with selenium. This observation could result from one of two different possibilities. Increased selenium availability could stabilize both isoforms, with methylation continuing at the same rate, resulting in a shift in the population of tRNA[Ser]Sec toward the methylated form. Alternatively, the methylated isoform may have greater inherent stability than its unmethylated precursor. The modification of the wobble nucleoside from mcm5U to mcm5Um is accompanied by a change in the conformation of tRNA[Ser]Sec (8
), which may contribute to differences in the stability of the two isoforms. Additional studies are underway to investigate which of these possibilities may be occurring.
Increased tRNA[Ser]Sec gene copy number has been shown to result in elevated tRNA[Ser]Sec levels in both cultured cells (11
) and transgenic animals (12
). In both of these studies, primer extension showed that increased tRNA[Ser]Sec levels achieved after transfection of the corresponding gene were accompanied by decreased levels of the native tRNA[Ser]Sec. By examining tRNA[Ser]Sec decay in cells that overexpress tRNA[Ser]Sec in this study, both with and without additional selenium supplementation, we determined that decay is accelerated in cells that overexpress the tRNA compared with vector-only transfected cells. These observations are consistent with the idea of a feedback mechanism that limits tRNA[Ser]Sec levels via RNA decay.
The results reported here contribute to an increasingly complex picture of the regulation of selenoprotein biosynthesis. Selenoproteins, such as members of the glutathione peroxidase, thioredoxin reductase and iodothyronine deiodinase families are likely to be important for many of the health benefits associated with dietary consumption of selenium. In this regard, it is important to note that tissue culture media are generally considered to be deficient in selenium compared with human serum (28
,29
), and the addition of selenium to culture media may be considered an appropriate model for the transition from a deficient to an adequate nutritional level. Collectively, the findings described here indicate that in addition to selenoprotein mRNA stability (30
) and limiting amounts of translational factors such as SBP2 (31
), the synthesis of selenoproteins and their regulation by selenium may be regulated by the stability of selenocysteine tRNA.
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FOOTNOTES |
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2 These authors contributed equally to this publication.
4 Abbreviations used: Act D, actinomycin D; CHO, Chinese hamster ovary; FBS, fetal bovine serum; GPx, glutathione peroxidase; mcm5U, methylcarboxymethyl-5'-uridine; mcm5Um, methylcarboxymethyl-5'-uridine-2'-O-methylribose; MEM, modified Eagle’s medium; Pol III, DNA Polymerase III; SBP2, SECIS element binding protein 2; SECIS, selenocysteine insertion sequence; TCA, trichloroacetic acid; tRNASer, serine transfer RNA; tRNA[Ser]Sec, selenocysteine transfer RNA.
Manuscript received 31 October 2001. Initial review completed 18 December 2001. Revision accepted 29 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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