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J. Biol. Chem., Vol. 275, Issue 36, 28110-28119, September 8, 2000
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From the
Received for publication, February 15, 2000, and in revised form, May 9, 2000
A common posttranscriptional modification of tRNA
is the isopentenylation of adenosine at position 37, creating
isopentenyladenosine (i6A). The role of this modified
nucleoside in protein synthesis of higher eukaryotes is not well
understood. Selenocysteyl (Sec) tRNA (tRNA[Ser]Sec)
decodes specific UGA codons and contains i6A. To address
the role of the modified nucleoside in this tRNA, we constructed a
site-specific mutation, which eliminates the site of isopentenylation,
in the Xenopus tRNA[Ser]Sec gene.
Transfection of the mutant tRNA[Ser]Sec gene resulted in
80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was
observed when transfected cells were treated with lovastatin, an
inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I
deiodinase protein. Furthermore, isopentenylation of
tRNA[Ser]Sec was not required for synthesis of Sec
on the tRNA. We conclude that isopentenylation of
tRNA[Ser]Sec is required for efficient translational
decoding of UGA and synthesis of selenoproteins.
Nucleoside modifications involving tRNAs at positions in or near
the anticodon have been shown to affect any of a number of steps in the
translation process (1). A tRNA position commonly modified is position
37, the site immediately 3' of the anticodon. Eukaryotic and bacterial
tRNAs reading codons starting with uracil often have a large
hydrophobic moiety attached to position 37 (2). One common modification
is isopentenylation of the adenosine at this position, creating
isopentenyladenosine
(i6A).1
Isopentenyl pyrophosphate:tRNA isopentenyltransferase is responsible for isopentenylation of this adenosine (3). The genes for isopentenyl pyrophosphate:tRNA isopentenyltransferase have been isolated in yeast
and bacteria. The Escherichia coli gene is miaA
(4), the Saccharomyces cerevisiae gene is MOD5 (5), and the
Schizosaccharomyces pombe gene most likely is
sin1 (6). Roles for the i6A modification in
cellular metabolism have mainly been deduced from mutant strains of
yeast and bacteria lacking isopentenyl pyrophosphate:tRNA
isopentenyltransferase activity. E. coli miaA In E. coli, the i6A modification is not required
for attachment of an amino acid to tRNA by aminoacyl tRNA synthetases
(10). However, this modification does appear to be important for
efficient binding of aminoacyl-tRNA to ribosomes (11). Isopentenylation of position 37 in tRNA appears to stabilize the first position of the
codon-anticodon interaction and thereby assists in preventing first
position misreading (10). However, both in vitro (12) and
in vivo (13) evidence indicate that the i6A
modification increases third position misreading due to decreased proofreading. Together these results show that, in yeast and bacteria, isopentenylation at position 37 in tRNAs reading codons beginning with
uridine has important physiologic effects on translation efficiency,
especially with regard to tRNA suppression of stop codons.
Mammalian selenocysteine (Sec) tRNA[Ser]Sec also contains
i6A at position 37 (see Ref. 14 for review and Ref. 15 for
subsequent work). Compared with other mammalian tRNAs, this tRNA is
unique in that Sec biosynthesis occurs on the tRNA after it is
aminoacylated with serine, it is 90 nucleotides long, which is the
longest eukaryotic tRNA sequenced to date, and it contains only four
other modified nucleosides in addition to i6A whereas other
tRNAs contain many more modified bases (14). Sec
tRNA[Ser]Sec decodes UGA and inserts Sec into nascent
selenopolypeptides. Recognition of UGA as a Sec codon and not as a stop
codon requires a stem-loop structure or structures in the
3'-untranslated region of selenoprotein mRNAs as well as other
translational factors specific for Sec incorporation (16).
In this paper, we examined the role of i6A at position 37 of tRNA[Ser]Sec on selenoprotein biosynthesis in
vivo by generating Sec tRNAs lacking this modified base. Our
studies indicate that structural changes at position 37 affect the
decoding properties of the altered tRNA[Ser]Sec and its
ability to decode UGA and incorporate Sec into proteins. The effect of
altered Sec tRNA[Ser]Sec on selenoprotein expression is
not because of differences in aminoacylation of the tRNA, biosynthesis
of Sec on the tRNA, transcriptional events, protein degradation, or
selenoprotein translation initiation. Most likely, the presence of
i6A is important for efficient translation of Sec codons by
tRNA[Ser]Sec.
Materials--
Taq DNA polymerase,
isopropyl-1-thio- Cell Culture--
Stock cultures of CHO-K1 cells were maintained
at 37 °C in a humidified chamber with a 5% CO2
atmosphere in Ham's F-12 medium containing 5% (v/v) newborn calf
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 25 mM Hepes, pH 7.4 (H-NCS).
Site-directed Mutagenesis--
The construct (A37C-XTSS) was
created by site-directed mutagenesis using a 1.04-kilobase
EcoRI/BamHI fragment of the Xenopus tRNA[Ser]Sec gene (18) as a template. Site-directed
mutagenesis was carried out by polymerase chain reaction amplification
of the 1.04-kilobase EcoRI/BamHI fragment in 2 reaction steps. Initially, a fragment of A37C-XTSS was generated using
the KpnI-tailed 29-mer (5'-CGGGGTACCCCACATCCACTAACAAACAG-3') and the 19-mer (5'-GCTACAGGTGTGAAGCCTG-3') to yield a 348-bp product. Simultaneously, a partially overlapping fragment was generated using
the 20-mer (5'-GGTATGTAAGCGGCGATACG-3') and a PstI-tailed 29-mer (5'-AAACTGCAGCCTGGTTGTTATGGATACTG-3') to yield a 365-bp product.
A portion of each reaction was combined along with the KpnI-tailed 29-mer and PstI-tailed 29-mer and
amplified by polymerase chain reaction using temperatures of 94 °C
for denaturation, 50 °C for annealing, and 72 °C for extension.
The polymerase chain reaction product was purified on a 1% agarose
gel, cut with KpnI and PstI, and cloned into
pGEM-3zf(+). The mutation was verified by DNA sequencing (19) performed
by the Tufts University Department of Physiology Core Facility. A clone
(A37A-XTSS) lacking the A37C mutation in the tRNA[Ser]Sec
gene sequence was isolated, sequenced, and used as a control.
Transfecting CHO Cells--
For isolation of
tRNA[Ser]Sec fractions used in the codon binding studies,
CHO cells were transfected by electroporation using 50 µg of plasmid
DNA from either A37C-XTSS or the polymerase chain reaction-derived
nonmutant A37A-XTSS. Cells were harvested 4 days post-transfection and
stored at
For [75Se]selenoprotein synthesis and experiments
involving Western and Northern blotting, CHO cells were transfected,
unless indicated otherwise, using LipofectAMINE Plus (Life
Technologies, Inc.) reagent according to the manufacturer. Briefly, CHO
cells were seeded on day 0 in 6-well plates at a density of 8 × 105 cells/well in 1.5 ml of H-NCS. On day 1, the cells were
transfected using 6 µl each of LipofectAMINE and Plus reagent with
the indicated plasmids in serum- and antibiotic-free Ham's F12. After
a 3-h incubation period, an equivalent volume of Ham's F12 containing 10% (v/v) NCS, 200 units/ml penicillin, 200 mg/ml streptomycin, 4 mM glutamine was added. On day 2, the cells were refed with H-NCS. Further processing of cells is described in the respective figure legend.
Extraction and Isolation of
tRNA[Ser]Sec--
Transfer RNA was isolated by phenol
extraction of CHO cells transiently transfected with A37A- or
A37C-XTSS. The extract was loaded onto a DE-52 column and the tRNA
eluted in 1 M NaCl, 0.1 M Tris-HCl, pH 7.4. Total tRNA was deacylated by incubation in 1.8 M Tris-HCl,
pH 8.0, for 1 h at 37 °C. Total tRNA was aminoacylated with
[3H]serine under conditions of limiting tRNA and
chromatographed in the absence of Mg2+ (20) on a RPC-5
column (21). Aminoacylated tRNA was eluted and chromatographed on a
RPC-5 column in a linear gradient at pH 4.5 containing 10 mM sodium acetate, 1 mM EDTA, and 0.575-0.8 M NaCl for A37C-XTSS or 0.6-0.8 M NaCl for
A37A-XTSS. CHO cells transfected with A37A-XTSS and treated with 20 µM lovastatin were fractionated in a gradient of
0.575-0.825 M NaCl. Fractionated [3H]seryl-tRNA[Ser]Sec isoacceptors were
pooled and used in ribosome binding studies according to the procedure
of Nirenberg and Leder (22) as described (23).
To examine the intracellular
[75Se]selenocystenyl-tRNA[Ser]Sec
population, CHO cells were transfected with either A37A-XTSS or
A37C-XTSS DNA by electroporation and allowed to grow for 4 days in
H-NCS supplemented with 50 ng/ml Na2SeO3. The
cells were harvested, radiolabeled for 4 h with
[75Se]selenious acid as described previously (24) at a
concentration of 44 µCi/ml of (A37C-XTSS) or 56 µCi/ml (A37A-XTSS),
washed, and then frozen until used for RNA extraction and RPC-5
chromatography as described above.
[75Se]Selenoprotein Synthesis--
CHO cells were
seeded and transfected as described above. On day 3, the cells were
radiolabeled for 3 h in H-NCS containing 2 µCi/ml
[75Se]selenious acid unless otherwise indicated. Cells
were washed and harvested for protein determination (25). Laemmli (26) or Schagger (27) SDS-PAGE was performed as indicated. Polyacrylamide gels were processed for fluorography and quantification of radiolabel incorporation was accomplished using a PhosphorImager (Molecular Dynamics) and ImageQuant software (Molecular Dynamics).
Northern Hybridization--
Total cellular RNA was isolated
(28), electrophoresed on 1% agarose/formaldehyde gels (29),
transferred to GeneScreen Plus, and probed for type I deiodinase using
a 770-bp SmaI/NcoI fragment of the G21-D10
plasmid (30) in a standard hybridization buffer (1 M NaCl,
1% (w/v) SDS, 10% (w/v) dextran sulfate, and 25 mg/ml sheared salmon
sperm DNA). The blot was stripped and reprobed for GAPDH using the
full-length rat cDNA (31). Alternatively, for Northern blot
analyses of Sec tRNA, total RNA, or RNA subjected to
immunoprecipitation were separated by electrophoresis on 15% acrylamide, 4 M urea gels, transferred to GeneScreen Plus
and probed for Sec tRNA (15) using a 193-bp
AvaI-HindIII fragment of the human
tRNA[Ser]Sec gene (17) as probe.
Probes were radiolabeled for 2 h at 37 °C with
[
Dot blot analysis of tRNA[Ser]Sec was accomplished as
described previously (15). Immunoprecipitation of total RNA for
Northern blot analysis by anti-i6A serum (32) was performed
essentially as described (15).
Hybridizations were monitored by autoradiography at Western Blotting--
CHO cells were seeded and transfected as
described above. Cells were harvested for protein determination (25)
and SDS-PAGE (26) as described. Protein gels were transferred to
Protran (Schleicher and Schuell) as described previously (30).
Membranes were blocked with 5% (w/v) nonfat milk in TBS-Tween (20 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween
20). Rabbit antisera directed against a peptide in the N-terminal
region of type I deiodinase (30) was used at a 1:1000 dilution in
TBS-Tween containing 5% (w/v) nonfat milk. Alternatively, rabbit
antisera directed against One means of defining the physiological role of i6A on
tRNA[Ser]Sec is to eliminate the adenosine at position
37, the site of isopentenylation. As described under "Experimental
Procedures," we created an A37C mutation in the Xenopus
tRNA[Ser]Sec gene (A37C-XTSS) by site-directed
mutagenesis. Next, we transfected A37C-XTSS into CHO cells, isolated
total RNA, and analyzed for Sec tRNA expression by northern
hybridization with tRNA[Ser]Sec probe (Fig.
1A, lanes 1 and
2). This experiment shows that A37C-XTSS transfected cells
express a new Sec tRNA with faster mobility than the endogenous CHO Sec
tRNA. Upon quantification, we observed that the expression levels of
A37C-Sec tRNA exceeded the level of the endogenous CHO tRNA by almost
4-fold.
To determine if A37C-Sec tRNA was isopentenylated, we
immunoprecipitated total RNA from cells transfected with either empty vector or A37C-XTSS with antiserum prepared in rabbits immunized with
isopentenyladenosine (32) and performed Northern blots on the
immunoprectipates (lanes 3 and 4). Previous work
showed that these antibodies do not recognize unmodified adenosine
(32). Whereas the endogenous, CHO Sec tRNA was precipitated by
our anti-i6A antibodies in both samples, the A37C-Sec tRNA
(lane 4) previously observed (lane 2) was not.
These data indicated that transfection of A37C-XTTS results in
overexpression of a Sec tRNA lacking isopentenyladenosine.
We also determined whether lovastatin caused reduced levels of
isopentenyladenosine in Sec tRNAs (Fig. 1B). Lovastatin is a
potent, specific inhibitor of mevalonic acid biosynthesis (35), the
precursor for the isopentenyl moiety at position 37 of Sec tRNAs.
Lovastatin treatment of A37A-XTSS transfected cells did not seem to
affect either endogenous CHO or exogenous A37A-Sec tRNA expression
(lanes 1 and 2). However, the amount of
i6A-containing Sec tRNA was reduced ~50% in total RNA
from lovastatin-treated cells (compare lanes 3 and
4). The decreased anti-i6A immunoprecipitation
in lovastatin-treated cells is consistent with reduced isopentenyl
modification of the tRNAs.
To address the effect that A37C-Sec tRNA has on cells, we examined the
capability of A37C-XTSS transfected cells to decode UGA Sec codons.
Fig. 2 depicts the synthesis of
selenoproteins in CHO cells as measured by 75Se
incorporation. As seen in untransfected cells (lane 1),
there are several endogenously expressed proteins that contain
75Se. This pattern is consistent with what others have
found in similar labeling experiments (33, 34). Furthermore, we
analyzed selenoprotein synthesis in CHO cells transfected with plasmids containing cDNAs for type I deiodinase (G21-D10) and type II
deiodinase (hD2/SelP). Transfection with G21-D10 alone resulted in the
expression of deiodinase (compare lanes 1 and 2),
and co-transfection with the A37A-XTSS construct appeared to have
little or no effect on the level of type I deiodinase expression
(compare lanes 2 and 3). However, co-transfection
with the mutant A37C-XTSS construct resulted in a dramatic reduction in
type I deiodinase expression (lane 4). Although type II
deiodinase expression was not as strongly expressed (lanes 5 and 6) as the type I form (lanes 2 and
3), its expression was affected similarly by co-transfection
with the wild-type (lane 6) and the mutant
tRNA[Ser]Sec gene constructs (lane 7).
Inhibition of Selenoprotein Synthesis by Selenocysteine
tRNA[Ser]Sec Lacking Isopentenyladenosine*
§¶,
,§§¶¶
Tufts University School of Medicine,
Department of Physiology, Boston, Massachusetts 02111, Harvard
Medical School, Thyroid Division, Department of Medicine, Brigham and
Women's Hospital, Harvard Institutes of Medicine,
Boston, Massachusetts 02115, and the ** Basic Research Laboratory,
Division of Basic Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutants show decreased translation of the trp operon (7) and manifest a
significant decrease in their ability to read through the stop codons,
UAG and UGA, that is reduced by 75% and 60-fold, respectively,
compared with wild-type E. coli (8). In MOD5 mutants of
S. cerevisiae, the efficiency of a tyrosine inserting nonsense suppressor tRNA is reduced relative to that of the wild-type organism (9). In addition, lack of i6A leads to
inactivation of a suppressor tRNA in the antisuppressor strain
sin1 of S. pombe (6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, and
5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal)
were obtained from Fisher. [
-32P]dCTP (3000 Ci/mmol)
was obtained from NEN Life Science Products, [3H]serine
(32 Ci/mmol) from Amersham Pharmacia Biotech, and
[75Se]selenious acid (180 Ci/mmol) was from the Missouri
Research Reactor Facility. All restriction enzymes and the cloning
vectors pGEM-T and pGEM-3zf(+) were obtained from Promega. Tri
reagent® for the isolation of total RNA was obtained from Molecular
Research Center. Protein A linked to Sepharose CL-4B, rabbit
anti--galactosidase antibody,
N-acetyl-leu-leu-norleucinal (ALLN), and all tissue culture
reagents were obtained from Sigma. LipofectAMINE and Plus reagents were
obtained from Life Technologies, Inc. Enhanced chemiluminescence reagents and GeneScreen Plus were obtained from NEN Life Science Products. The plasmid pUCpST, containing a 193-bp
AvaI-HindIII fragment encoding the human
tRNA[Ser]Sec gene (17) ligated into pUC18, was kindly
provided by Dr. Alan Diamond (University of Illinois-Chicago). The
plasmid hD2/SelP, expressing type II deiodinase, was kindly provided by
Dr. Christoph Buettner (Harvard Medical School), the full-length rat
cDNA for GAPDH used in Northern blot analyses was provided by Dr.
Daniel Ortiz (Tufts Medical School), and lovastatin was from Merck.
80 °C until tRNA isolation.
-32P]dCTP using DECAprimeTM II (Ambion Inc.), added
to a final concentration of 0.1 nM, and incubated at
62 °C for a minimum of 16 h. Following hybridization, the
membranes were washed for 2 h in 1% (w/v) SDS, 0.1× SSC at
62 °C with multiple changes of the wash buffer.
80 °C in the
presence of intensifying screens, and quantification was accomplished
using a PhosphorImager (Molecular Dynamics) and ImageQuant software
(Molecular Dynamics).
-galactosidase was used at a 1:1000
dilution. Goat anti-rabbit horseradish peroxidase was used as the
secondary antibody at a 1:4000 dilution in TBS-Tween containing 5%
(w/v) nonfat milk. Enhanced chemiluminescence was performed according
to the manufacturer (NEN Life Science Products).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 1.
Expression and isopententylation of
Xenopus A37A- and A37C-Sec tRNA in CHO cells. In
A, CHO cells were transfected with pGEM-T vector alone
(lanes 1 and 3) or A37C-XTSS (lanes 2 and 4) via either LipofectAMINE (lanes 1 and
2) or electroporation (lanes 3 and 4)
as described under "Experimental Procedures." Total cellular RNA
from each sample was prepared and analyzed by Northern blotting for Sec
tRNA expression (lanes 1 and 2). Alternatively,
20 (lane 3) or 30 µg (lane 4) of total cellular
RNA from electroporated cells were immunoprecipitated with
anti-i6A serum, and the immunoprecipitates analyzed by
Northern blotting for isopententylated Sec tRNA expression as described
previously (15). In B, all cells were
LipofectAMINE-transfected with A37A-XTSS and incubated in the absence
(lanes 1 and 3) or presence (lanes 2 and 4) of 10 µM lovastatin for 18 h.
Total RNA was prepared, and 20 µg were blotted for Northern blot
analysis directly (lanes 1 and 2). Also, 40 µg
of total RNA was subjected to anti-i6A immunoprecipitation
(15) prior to Northern blot analysis (lanes 3 and
4). Northern blot analysis and quantification of Sec tRNA
expression were performed as described under "Experimental
Procedures."
View larger version (49K):
[in a new window]
Fig. 2.
A37C-Sec tRNA expression reduces deiodinase
synthesis in CHO cells. CHO cells were transfected with vector
(pGEM-T) alone, type I (G21-D10), or type II (hD2/SelP) deiodinase
plasmid DNA alone or in combination with A37A-XTSS or the mutant
A37C-XTSS gene as described under "Experimental Procedures." On day
3, CHO cells were pulsed for 3 h with 2 µCi/ml of
75Se in H-NCS. The cells were harvested, an extract was
prepared, and 100 µg of cell protein were electrophoresed on 13%
SDS-PAGE gels. Gels were processed for fluorography, and quantification
of [75Se]selenoprotein synthesis was accomplished by
Phosphorimage analysis using ImageQuant software. This represents a
typical fluorogram of 75Se incorporation into type I and
type II deiodinase. Lane 1, control, 1.6 µg of pGEM-T DNA;
lane 2, 0.4 µg of G21-D10 DNA + 1.2 µg of pGEM-T DNA;
lane 3, 0.4 µg of G21-D10 DNA + 1.2 µg of A37A-XTSS DNA;
lane 4, 0.4 µg G21-D10 DNA + 1.2 µg A37C-XTSS DNA;
lane 5, 0.4 µg of hD2/SelP + 1.2 µg of pGEM-T DNA;
lane 6, 0.4 µg of hD2/SelP + 1.2 µg of A37A-XTSS DNA;
and lane 7, 0.4 µg of hD2/SelP + 1.2 µg of A37C-XTSS
DNA.
Quantification of exogenously derived deiodinases and endogenous selenoprotein synthesis is shown in Table I. Expression of wild type A37A-XTSS gene had variable, but marginal affects on the expression of the deiodinases when compared with cells transfected with either G21-D10 or hD2/SelP alone. However, A37C-XTSS co-transfection with either deiodinase gene resulted in 78 and 94% decreases in the syntheses of type I and type II deiodinases, respectively. Whereas, expression of the wild type, Xenopus A37A-XTSS construct produced a slight increase in endogenous selenoprotein biosynthesis, the mutant A37C-XTSS gene led to a slight decrease. Synthesis of thioredoxin reductase (~54 kDa) and the 24-and 18-kDa selenoproteins were reduced between 9 and 26% by A37C-Sec tRNA expression. The difference in the effect of A37C-Sec tRNA expression seen between the deiodinase proteins and other endogenous selenoproteins may be related to transfection or other translation phenomena and is addressed under "Discussion." Neither result could be explained by decreased cellular uptake of 75Se. Uptake levels of 75Se, when normalized for cell protein, were not affected by experimental treatment (data not shown).
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Although the effect was not as dramatic as in CHO cells, co-transfection of A37C-XTSS along with the deiodinase constructs into HEK-293 cells led to decreased syntheses of both type I and type II deiodinases (data not shown). It should be noted that [75Se]selenious acid incorporation into either type I or II deiodinase was not detected in either CHO (Fig. 2) or HEK-293 (data not shown) cells transfected with pGEM-T vector alone.
To determine if the effect of A37C-XTSS was because of a general
inhibition of exogenous gene expression, we transfected CHO cells with
either G21-D10 or the -galactosidase encoding plasmid DNA,
pNASS, in the presence or absence of either A37A-XTSS or A37C-XTSS and performed Western blot analyses. As seen in Fig. 3, transfection of A37C-XTSS reduced type
I deiodinase levels (compare lanes 2 and 4) to a
similar extent as that seen in Fig. 2. However, -galactosidase
expression was unaffected by co-expression of A37C-Sec tRNA (compare
lanes 5 and 7). These data indicate that A37C-Sec
tRNA does not act as a general inhibitor of protein synthesis, but
inhibits selenoprotein expression (note that the immunoreactive protein
seen at ~21 kDa represents nonspecific binding of our antiserum).
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In the above experiments, the function of i6A was addressed by creating a mutation in the tRNA gene sequence, transfecting this mutant construct into CHO cells, and examining the ability of cells transfected with the mutant Sec tRNA gene to support selenoprotein synthesis. This method addressed the function of the intact, modified nucleoside at position 37 of Sec tRNA. As a second approach to discern the function of the isopentenyl moiety, we treated cells with 10 µM lovastatin. Lovastatin is a potent, specific inhibitor of mevalonic acid biosynthesis (35), and mevalonic acid is a precursor for the isopentenyl moiety at position 37 of tRNA. As observed above (Fig. 1C), lovastatin treatment of cells expressing A37A-Sec tRNA resulted in an ~50% decrease in the amount of i6A-containing Sec tRNA without affecting total Sec tRNA expression. This experimental protocol allowed us to further address the function of the isopentenyl modification on selenoprotein biosynthesis.
CHO cells were transfected with G21-D10 plasmid DNA and subsequently
cultured for the final 21 h under a variety of conditions in the
presence or absence of lovastatin. [75Se]Selenious acid
was added during the final 3 h to monitor selenoprotein synthesis.
Experiments were carried out in duplicate (Fig.
4), and the resulting data were
quantified (Table II). The pattern of
selenoprotein labeling in the absence of lovastatin is shown in
lanes 1 and 2. In the presence of 10 µM lovastatin, selenoprotein labeling was dramatically
reduced (lanes 3 and 4), whereas the addition of
mevalonic acid to the culture medium reversed the effect of
lovastatin (lanes 5 and 6). Quantifying this data
showed that all cellular selenoproteins were reduced from 55 to 95%
with the exception of an 8.5-kDa protein. The presence of the 8.5-kDa protein is enhanced, and its identity is unknown.
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It has previously been shown that 10 µM lovastatin results in cell-cycle arrest at G0/G1 phase (32). To ascertain how much of the lovastatin effect was because of cell cycle arrest, we cultured cells under serum-free conditions. CHO cells cultured for 21 h under serum-free conditions (lanes 7 and 8) showed reduced 75Se incorporation into several selenoproteins compared with CHO cells cultured in H-NCS (lanes 1 and 2). The latter growth conditions prevent cell cycle arrest. The magnitude of inhibition, however, was much less than that observed with lovastatin treatment. Note that 75Se incorporation into type I deiodinase (~27 kDa) was unaffected by serum removal. CHO cells cultured under serum-free conditions in the presence of 10 µM lovastatin showed a further reduction in 75Se incorporation into all proteins, except the 8.5-kDa protein (lanes 9 and 10). Most notably affected was type I deiodinase, whose synthesis was unaffected by serum-free conditions.
Together these data point to a crucial role of the isopentenyl group at position 37 of the Sec tRNA in selenoprotein expression. However, the decreased synthesis of type I deiodinase may be explained by lower levels of deiodinase mRNA, a decrease in protein stability, or a reduction in mRNA translation.
First, we used Northern blot analysis to determine whether the type I
deiodinase mRNA levels were reduced in A37C-XTSS-transfected or
lovastatin-treated CHO cells. As shown in Fig.
5, type I deiodinase mRNA is
undetectable in the absence of G21-D10 transfection (lane 1). When normalized for GAPDH expression, neither A37C-XTSS
transfection (lane 4) nor lovastatin treatment (lane
5) affected the amount of type I deiodinase mRNA when compared
with cells transfected with G21-D10 alone (lane 2).
Interestingly, the ratio of type I deiodinase to GAPDH in cells subject
to serum deprivation (lane 6) was similar to that observed
under the other conditions (lanes 2-5). These results
indicate that the manipulations that cause a decreased synthesis of
type I deiodinase do not alter rates of gene transcription or mRNA
stability. This implicates a post-transcriptional event as the cause
for the observed reduction in selenoprotein synthesis by the mutant
A37C-Sec tRNA and the lovastatin-induced Sec tRNA lacking
i6A.
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Next, we performed a pulse-chase experiment to determine if
co-transfection with the mutant A37C-XTSS construct affected the degradation rate of type I deiodinase protein (Fig.
6). CHO cells were co-transfected with
G21-D10 and either A37A-XTSS (lanes 1-4) or A37C-XTSS
(lanes 5-8). After one day, the cells were pulse-labeled with 75Se for 1.5 h and chased for various times, and
the amount of radiolabeled type I deiodinase was determined after
SDS-PAGE and fluorography. Densitometric scans showed that
A37C-XTSS-transfected cells synthesized ~35% less type I deiodinase
during the pulse interval compared with A37A-XTSS-transfected cells.
However, the rate of disappearance of type I deiodinase was similar in
both sets of cells. These data indicate the expression of the mutant
tRNA does not affect the stability of type I deiodinase.
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Lastly, we sought to determine if A37C-XTSS inhibited type I deiodinase expression by decreasing translation. Inhibition of translation could occur at two possible sites: initiation of synthesis or, given the unique nature of selenoprotein biosynthesis, decoding of the internal Sec UGA. Impaired read through of the internal UGA would cause termination of translation and release of a truncated protein.
Fig. 7 is a Western blot analysis showing
the level of both the full-length and truncated type I deiodinase in
the absence (lanes 1 and 3) or presence
(lanes 2 and 4) of co-transfected A37C-XTSS. We
also analyzed the content in cells treated with ALLN, an inhibitor of
cellular proteolysis (lanes 3 and 4). The approximate relative expression of the full-length protein in lanes 1-4 is 1.0, 0.35, 0.15, and 0.06, respectively.
|
If A37C-Sec tRNA caused decreased initiation of translation, we would expect to observe similar reductions in both the full-length and truncated type I deiodinase proteins. On the other hand, if the mutant Sec tRNA inhibited expression of the full-length type I deiodinase by blocking decoding of the internal UGA, we would observe a reciprocal increase in the amount of truncated protein. In comparing lanes 1 and 2, we see an ~3-fold reduction in the amount of full-length protein, yet no change in the expression of the truncated translate. This observation does not support either mechanism for A37C-Sec tRNA inhibition of translation.
We then looked at the effect of ALLN on the expression of type I deiodinase proteins (Fig. 7, lanes 3 and 4). ALLN caused a dramatic increase in the amount of the truncated protein. This observation indicates the truncated protein is subject to extremely rapid degradation, probably as a result of the proteosome-dependent protease system (36). Given the abundance of truncated versus full-length protein in the presence of ALLN, we conclude that the overwhelming majority (~95%) of type I deiodinase mRNA-directed translation is terminated at the internal UGA. Similarly, synthesis of the full-length protein is extremely inefficient (~5% of total mRNA-directed translation).
Unfortunately, this grossly disproportional pattern of expression for the two forms of type I deiodinase prevents us from determining if A37C-Sec tRNA directly inhibits decoding of the internal UGA. Co-transfection of the mutant tRNA gene reduces full-length deiodinase expression by ~60% in ALLN-treated cells. However, (as noted above) only ~5% of mRNA-directed translation produces the full-length protein. Therefore, if the mutant tRNA does inhibit decoding, we would expect to observe only an ~3% (60% of 5%) increase in the amount of truncated protein. Such a small difference would not be detected in the immunoblots from ALLN-treated cells.
If the initiation of translation is inhibited, we would predict a 60% decline in truncated deiodinase expression in the ALLN-treated cells. However, densitometric scans of films with reduced exposures show no diminution in the amount of the truncated protein in ALLN-treated cells (data not shown). These data solidify our conclusion (from lanes 1 and 2) that the mutant tRNA does not inhibit initiation of translation.
Another possible explanation for the reduction in 75Se
incorporation into protein seen with lovastatin treatment and A37C-XTSS expression could be because of the lack of or reduced
selenocystenyl-tRNA[Ser]Sec biosynthesis because of
changes at position 37. Fig. 8 shows the
RPC-5 column elution profiles of
[75Se]selenocystenyl-tRNA[Ser]Sec from CHO
cells transfected with either A37A-XTSS (Fig. 8A) or A37C-XTSS (Fig. 8B). The elution profiles of both in
vivo 75Se-labeled tRNA (Fig. 8A) and
in vitro labeled [3H]seryl-tRNA (Fig.
8C) from CHO cells overexpressing A37A-XTSS show two peaks;
the position 34, 5-methylcarboxymethyluridine (mcm5U, peak
II) and 5-methylcarboxymethyluridine-2'-O-methylribose tRNAs[Ser]Sec (mcm5Um, peak III)
as previously reported (37). Peak Ic observed in Fig.
8B is most likely the A37C-Sec-tRNA given its relatively early elution from the RPC-5 column as was seen for
tRNA[Ser]Sec lacking i6A (38). Similarly peak
Ia in Fig. 8D isolated from cells overexpressing A37A-XTSS and subsequently treated with 20 µM lovastatin
most certainly lacks i6A. Therefore it can be concluded
that i6A is not necessary for aminoacylation with serine or
in the biosynthesis of Sec.
|
The efficiency of aminoacylation of tRNA[Ser]Sec and biosynthesis of Sec were still unknown. Measurement of these parameters was accomplished by quantifying the relative amount of tRNA[Ser]Sec in each of these peaks by dot blotting and the amount of 75Se associated with the purified Sec tRNA by liquid scintillation counting. The calculated specific radioactivity (Table III) is a measure of the ability of the tRNA to become aminoacylated with Sec. Experiment 1 shows the specific activities of the two wild type forms of Sec-tRNA (derived from peaks II and III, Fig. 8A) from cells transfected with A37A-XTSS. Experiment 2 compares the purified mutant, A37C-Sec-tRNA (peak Ic, Fig. 8B), with the two forms of A37A-Sec-tRNA (peaks II and III, Fig. 8B). Note, the specific radioactivity is approximately the same for all three Sec-tRNA species. These data indicate that the mutant Sec tRNA is aminoacylated with [75Se]Sec to a similar extent as A37A-Sec tRNA. Thus, a lack of or a reduction in aminoacylation of A37C-Sec tRNA or in the biosynthesis of Sec cannot explain the observed decreases in selenoprotein synthesis seen in Figs. 2 and 3.
|
To determine if tRNA[Ser]Sec minus i6A may have an effect at the level of translation, we examined the codon recognition properties of the mutant isoacceptor and the i6A lacking isoacceptor induced by lovastatin inhibition. As shown in experiments 1 and 2 in Table IV, changes at position 37 from i6A37 to either A37C or unmodified A37A did not alter the specificity of the seryl-tRNA[Ser]Sec for recognizing UGA in a ribosome binding assay. The cysteine codons, UGU and UGC, as well as the tryptophan codon, UGG, were poorly recognized by all seryl-tRNA[Ser]Sec isoacceptors tested in this assay. However, when we examined specific binding as a percentage of added seryl-tRNA[Ser]Sec for A37C- and i6-lacking seryl-tRNA[Ser]Sec to UGA in this assay, both tRNAs exhibited reduced binding to UGA in comparison to the wild-type isoacceptor. Specific binding of the A37C- and i6-lacking Sec tRNA to UGA was decreased by 62 and 69%, respectively.
|
Although E. coli ribosomes were used in the ribosomal
binding studies, it should be noted that the genetic language of
mammals and bacteria were shown to be the same in this assay using
E. coli ribosomes (39) and that strong responses relative to
their assigned codons of mammalian Cys-, Arg-, and Trp-tRNAs to UGA were observed in this same assay using E. coli ribosomes
(40). The later observation suggests that mammalian Cys-, Arg-, and Trp-tRNAs are capable of misreading UGA and, indeed, these tRNAs serve
as authentic suppressors of UGA stop codons in mammals (41, 42). We
conclude, therefore, that the data shown in Table IV demonstrate a
defect in the decoding properties of tRNA[Ser]Sec lacking
the i6A modification in mammalian protein synthesis.
| |
DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study we have provided evidence that changes at position 37 of Sec tRNA result in a tRNA with reduced abilities to bind to UGA in an in vitro ribosomal binding assay and to support selenoprotein synthesis in vivo. These results elucidate the role of i6A in mammalian cells that seems to be similar to that observed in bacteria and yeast. In E. coli (8) and S. cerevisiae (9), the inability to synthesize i6A results in a decreased efficiency to suppress stop codons. Similarly, the lack of i6A on a nonsense serine suppressor, sup3-i, in an antisuppressor mutant of S. pombe, sin1, leads to its inactivation (6). In addition to the decreased stop codon suppression seen in bacteria and yeast, we have shown that removal of the i6A from Sec tRNA results in reduced selenoprotein synthesis in mammalian cells.
Our observations of reduced selenoprotein synthesis are quite intriguing. First, the effect of A37C-XTSS transfection on type I deiodinase synthesis (Table I) does not mimic the effect on endogenous selenoprotein synthesis. Whereas a definitive effect of A37C-Sec tRNA expression on endogenous selenoproteins was observed, the reduction in synthetic rates did not approach the 80-90% decreases observed with type I and II deiodinases. Attempts to differentiate between a unique effect on deiodinase and a general effect on transfected selenoprotein cDNAs was attempted using HepG2 cells. This cell line reportedly expresses functional type I deiodinase activity (43). However, we were unable to observe detectable 75Se-radiolabeling of type I deiodinase under our culture conditions indicating that the synthetic rate is too low for determining the effect of A37C-Sec tRNA on endogenous type I deiodinase synthesis.
Why does expression of the mutant, A37C-tRNA[Ser]Sec appear to preferentially inhibit deiodinase synthesis? A plausible explanation is that replacing the highly modified i6A nucleoside with an unmodified base results in an isoacceptor that, by analogy to the corresponding tRNAs in bacteria and yeast, decodes UGA less efficiently than the fully modified form. Our co-transfections were performed with the aid of liposomes, and the mutant tRNA and cDNA plasmids were mixed prior to the addition of the liposomes. Under these conditions, the mixture of exogenous DNA forms complexes with the liposomes. It is highly likely both types of DNA are present in every complex. Therefore, those cells that take up a liposome-DNA complex are receiving both types of plasmid, and it is unlikely a significant amount of cells take up one type of DNA without the other. Also, knowing that transfection efficiency is not 100%, some percentage of the population is not receiving any mutant tRNA. However, the entire population is exposed to a radiolabeled 75Se precursor. Consequently, if the mutant tRNA were inhibiting endogenous selenoprotein synthesis, we would only observe a decline in radiolabeled incorporation equivalent to the percentage of cells expressing the mutant tRNA. As seen in Table I, we observed a small, but significant decline (9-26%) in the incorporation of 75Se into several endogenous selenoproteins when transfected with the mutant tRNA. On the other hand, all cells on the monolayer receive lovastatin and are inhibited by the drug. This may explain why endogenous selenoproteins and transfected type I deiodinase syntheses were inhibited by the drug.
Of course other possibilities exist to explain the lack of effect of
A37C-Sec tRNA on endogenous selenoprotein synthesis. For example,
several laboratories have provided evidence for the existence of
supramolecular translation complexes containing mRNAs, ribosomes,
EF-1, tRNAs, and tRNA synthetases (44-46). According to the
channeling model of Stapolionis and Deutscher (46), tRNAs are directly
transferred from aminoacyl-tRNA synthetases to elongation factor to
ribosomes without dissociation into the cellular fluid. Upon leaving
the ribosome, uncharged tRNAs are directly transferred back to their
cognate synthetases, without being released into the surrounding
cytosol. There, they are recharged with amino acid and then bound by
EF-1 for another elongation cycle in the same supramolecular
complex. This model may explain the effect of transfected Sec-specific
tRNA on transfected but not endogenous selenoprotein synthesis.
Selenoprotein mRNAs expressed from endogenous genes would be expected to be continuously transcribed and exported to the cytoplasm at a low, constitutive level. Newly synthesized selenoprotein mRNA and Sec tRNA from the transfected genes would likely undergo a sharp, steep burst of transcription and export. Thus, most of the endogenous selenoprotein mRNAs would already be in the cytoplasm assembled into translation complexes at the time of transfection. The newly transcribed mRNAs, following export, would predominantly be free to assemble together into new supramolecular complexes, producing an apparent transfection-specific effect of the introduced wild-type or mutant tRNA[Ser]Sec gene.
As was shown in Fig. 5, no effect of A37C-XTSS co-expression or lovastatin treatment on type I deiodinase mRNA levels was observed. Whereas increased selenoprotein degradation could account for some of the decreased 75Se-labeled proteins in Fig. 2 (lanes 4 and 7) and Fig. 4 (lanes 3 and 4), we hypothesized that A37C-Sec tRNA and i6A-lacking Sec tRNA affect translation. To address this, we examined the ability of these tRNAs to recognize UGA in a well established ribosomal binding assay (22). The results in Table IV provide evidence that changes at position 37 of the Sec tRNA affect translation, specifically the step involved in codon binding. Specific binding to UGA was reduced ~70% for both the A37C- and i6A-lacking tRNA[Ser]Sec populations compared with the corresponding wild-type tRNA. These results are in close agreement to those of Ohama et al. (47). They observed that an early eluting 75Se-labeled tRNA showed a 50% decrease in specific binding to UGA compared with the full modified Sec tRNA. They presumed this to be a Sec-tRNA lacking i6A based on previous observations in Xenopus oocytes (38).
Our hope was to test the ability of these individual Sec tRNA populations to support UGA translation in vitro. Rabbit reticulocyte translation systems programmed with specific mRNAs are widely used for in vitro synthesis of proteins. However, to our knowledge an in vitro translation system to faithfully measure the efficiency of selenoprotein synthesis does not exist. Previously, Berry et al. (30, 48) demonstrated in vitro synthesis of type I deiodinase using a rabbit reticulocyte system. However, these investigators determined that the commercially available in vitro translation systems are, in addition to being quite inefficient, a measure of UGA suppression and not Sec incorporation into protein. Given that the A37C- and i6A-lacking tRNA[Ser]Sec both exhibit binding to UGA, we would expect them to show some level of UGA translation. However, without a reliable system to accurately measure translation efficiency, we are unable to definitively demonstrate a reduced translational ability of either the A37C- or i6A-lacking tRNA[Ser]Sec.
We are not aware of prior studies addressing the specific role of
adenosine adjacent to the anticodon in any tRNA. In addition to
preventing isopentenylation, inserting a cytosine for adenosine at
position 37 in the Sec tRNA may have other effects on its function, e.g. inhibiting other modifications, affecting tRNA
stability, altering tertiary structure, and inhibiting general protein
synthesis. However, our data show the lack of adenosine at this
position does not affect the ability of A37C-Sec tRNA to bind Sec, as
judged by normal amounts of 75Se incorporation into the
mutant tRNA (Fig. 8B and Table III). This observation
indicates adenosine 37 is not required for serine-specific aminoacylation and tRNA-dependent conversion to Sec,
mediated by aminoacyl-tRNA synthetase and selenocysteine synthase,
respectively. Furthermore exchanging guanosine for adenosine at
position 37 of Sec tRNA does not affect the tertiary interaction of
D-T-arms in the tRNA.2
Lastly, the expression of transfected -galactosidase is not reduced
by co-transfection with the A37C-Sec tRNA gene (Fig. 3), showing lack
of adenosine at position 37 does not grossly affect general protein synthesis.
These data provide evidence of a crucial role for the i6A
modification on tRNA[Ser]Sec for decoding UGA Sec codons.
Future directions are aimed at determining the precise mechanism by
which changes at position 37 of Sec tRNA result in decreased
selenoprotein synthesis.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Alan Diamond (University of Illinois-Chicago) and Laura Liscum (Tufts University) for helpful discussions. We also thank Heather Charles for excellent technical assistance and Dr. Byeong Jae Lee (Seoul National University) for help with the mutant tRNA[Ser]Sec construction.
| |
FOOTNOTES |
|---|
* This work was supported by Grant MCB-9316131 from the National Science Foundation (to J. R. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Enanta Pharmaceuticals, 750 Main St., Cambridge, MA, 02139.
¶ Supported by National Institutes of Health Training Grant T32 DK07542.
§§ To whom correspondence should be addressed: Tufts University School of Medicine, Dept. of Physiology, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-2405; Fax: 617-636-0445; E-mail: jerry.faust@ tufts.edu.
¶¶ Established Investigator of the American Heart Association.
Published, JBC Papers in Press, May 19, 2000, DOI 10.1074/jbc.M001280200
2 B. J. Lee, personal communication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: i6A, isopentenyladenosine; Sec, selenocysteine; ALLN, N-acetyl-leu-leu-norleucinal; bp, base pairs; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; mcm5U, 5-methylcarboxymethyluridine; mcm5Um, 5-methylcarboxymethyluridine-2'-O-methylribose.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
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