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INTRODUCTION |
The yeast GSPT gene, whose product is a GTP-binding
protein structurally related to the translation elongation factor
EF1
, was first isolated based on the ability to complement a
temperature-sensitive gst1 mutation of Saccharomyces
cerevisiae (1). Because DNA synthesis was substantially arrested
at the nonpermissive temperature in this mutant, the GSPT
gene product appears to play an essential role at the G1 to
S phase transition in the yeast cell cycle. On the other hand,
SUP35 was cloned by another group investigating omnipotent
suppressor mutant of S. cerevisiae, and the gene turned out
to be identical to GSPT (2). Omnipotent suppressor is a class of nonsense suppressors that is recessive and effective toward
three types of nonsense codons (3). Mutations in the GSPT/SUP35 gene were shown to increase the level of
translational ambiguity (4, 5), suggesting that this gene product may also function as a positive regulator of translational accuracy in yeast.
In eukaryotic protein synthesis, all three types of termination codons
are directly recognized by a polypeptide chain releasing factor, eRF1,
to release synthesized polypeptide chain from ribosome, and another
releasing factor, eRF3, appears to be essentially required for the
ribosomal binding of eRF1. Recently, it was reported in S. cerevisiae (6) and Xenopus laevis (7) that the product of the GSPT/SUP35 gene forms a complex with eRF1 to function
as eRF3. We previously cloned a human homologue of the yeast
GSPT gene (8) and more recently isolated two mouse GSPT
genes, the counterpart of human GSPT1 and a novel member of the GSPT
gene family, GSPT2 (9). The mammalian GSPT1 and GSPT2 could also associate with eRF1 to function as eRF3, although the two GSPTs were
clearly distinct from each other in terms of their amino-terminal sequences, the expression during cell cycle progression, and tissue distribution (9). It thus appears that there is a functional conservation of this family from yeast to mammals in terms of translation termination. In this report, we present evidence that GSPTs
may have another function by interacting with PABP that binds to the
3'-poly(A) tail of eukaryotic mRNAs. Our present results indicate
that GSPT/eRF3 may play important roles not only in the termination of
protein synthesis but also in the regulation of mRNA stability.
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EXPERIMENTAL PROCEDURES |
Screening of Yeast Two-hybrid Library--
A yeast two-hybrid
screen was performed according to the standard method of Fields and
Song (10) utilizing the Matchmaker Two-hybrid System
(CLONTECH). A human T cell lymphoma cDNA
library in the pACT vector was screened using the full-length mouse
GSPT1 inserted into pGBT9 as a bait. The library and bait were
cotransformed into the yeast two-hybrid strain HF7c using a standard
lithium acetate-polyethylene glycol method. A total of 6.5 × 106 individual recombinant clones were screened. Positive
clones grown on His
medium were selected for activation
of HIS3 reporter gene, and
-galactosidase assay was performed as
described previously (9, 11). A series of deletion mutants of GSPTs and
PABP1 I were constructed
using a PCR-based strategy. PCR products encoding amino- and
carboxyl-terminal truncations of GSPTs and PABP I were subcloned into
pGBT9 in-frame with the GAL4 DNA-binding domain. The two-hybrid vectors
thus obtained were cotransformed into the yeast host strain SFY526.
Transformed colonies were replated on tryptophan- and leucine-deficient medium.
Production of Recombinant Proteins--
For production of
GST-fused PABP, a full-length human PABP I cDNA was exised from
yeast two-hybrid positive clone (pGAD10:PABP) and directionally
subcloned into pGEX6P1 vector (Amersham Pharmacia Biotech) downstream
of the glutathione S-transferase (GST) gene. For the
GST-fused amino-terminal domain (amino acids 1-204) of GSPT2, PCR
products encoding the corresponding sequences were subcloned into
pGEX6P1 in-frame with the GST. The GST-fused proteins and GST alone
were expressed in Escherichia coli HB101 following induction
by 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside at 20 °C for
9 h. The expressed proteins were recovered from the cells and
resuspended in TEDN buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40)
containing protein inhibitors (1 µg/ml of leupeptins, 1 µg/ml of
pepstatin A, 2 µg/ml of aprotinin, and 100 µM
phenylmethylsulfonyl fluoride). Following incubation with 1 mg/ml of
lysozyme at 4 °C for 30 min, the cell lysates were sonicated for 3 min on ice. The extracts were centrifuged at 100,000 × g for 60 min, and the expressed proteins in the clear supernatant were purified using a glutathione-Sepharose column according to the manufacturer's instructions. For electrophoretic mobility shift assay, the GST portion of the expressed PABP was excised
using PreScission protease (Amersham Pharmacia Biotech) according to
the manufacturer's instructions.
For production of a recombinant GSPT1, the
NcoI-XhoI fragment of pTrcHis vector (Invitrogen,
Co) was first removed and annealed with the synthetic adaptor MCS
(MCS5' plus MCS3') as described below to make pTrc. The pTrc was then
digested with BamHI and PstI and replaced by the
BamHI-PstI portion of the pGH5, which contained
the full length of human GSPT1 cDNA sequence (9). The GSPT1
expression vector (pTrc:GSPT1) thus obtained was introduced into
E. coli strain JM109 to produce the recombinant GSPT1. The bacterial cells were cultured at 37 °C with vigorous shalking in 1 liter of an LB medium containing 0.2% glucose. After the culture
reached the density of 0.4 A600, GSPT1 was
induced by the addition of 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside. After an
additional 2-h culture, the cells were harvested by centrifugation at
4,000 × g for 25 min. The cells were resuspended in
TEDN and processed as described above for the preparation of the GST
fusion protein. The synthetic oligonucleotides used were:
MCS5'-CATGGATCCCGGGTCGACTCTAGAGGCC and
MCS3'-TCGAGGCCTCTAGAGTCGACCCGGGATC. FLAG-tagged mouse GSPT2 and its amino-terminal deletion mutants were produced in COS-7 cells as
described previously (9).
In Vitro Binding Assay--
The purified GST-fused PABP or GST
alone (0-10 µg) was incubated with E. coli extract
containing GSPT1 (approximately 10 µg) at 4 °C for 60 min in 200 µl of TEDN. Glutathione-Sepharose resin (20 µl) that had been
pretreated with 5% bovine serum albumin was added to the reaction
mixture and further incubated at 4 °C for 60 min with gentle mixing.
The resin was extensively washed three times with 500 µl of TEDN
containing the protein inhibitors and incubated with 20 µl of 20 mM glutathione at 4 °C for 30 min in 50 mM
Tris-HCl (pH 7.5). After centrifugation, 20 µl of the supernatant was
mixed with 20 µl of 2× SDS-polyacrylamide sample buffer and boiled
for 5 min, followed by SDS-polyacrylamide gel electrophoresis (12.5%
of acrylamide). Immunoblots were performed with rabbit antisera raised
against a recombinant human GSPT1, PABP, or GST as described previously
(9).
Gel Mobility Shift Assay--
The assay was performed as
described previously (15). Indicated amounts (up to 10 pmol) of the
purified PABP was first incubated with or without the GST-fused
amino-terminal domain (amino acids 1-204) of GSPT2 (40 pmol) in TEDN
at 4 °C for 30 min. The mixture was then incubated with
5'-32P-labeled poly(A) RNA (23-mers; 1 nM) at
4 °C for 30 min in a buffer (total volume, 20 µl) consisting of 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 0.01% acetylated bovine serum
albumin, 5.3 mM EDTA, 0.01% Nonidet P-40, and 100 mM
-mercaptoethanol. Half of the reaction mixture was
electrophoresed at 20 V/cm in 6% polyacrylamide gels (60:1; 1× TBE
and 0.1% Triton X-100) under nondenaturing conditions at 4 °C for
60 min. The radioactivity retained on the gel was visualized with a
Fuji BAS 2000 bioimaging analyzer.
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RESULTS AND DISCUSSION |
No Involvement of the Amino-terminal Domain of GSPT in the Binding
to eRF1--
In the previous study (9), we reported that mammalian
GSPT1 and GSPT2 could directly interact with eRF1 in the assays of yeast two-hybrid screening and in vitro binding. Because the
GSPT family consist of a conserved carboxyl-terminal region homologous to EF1
GTP-binding protein and an extra nonhomologous amino-terminal region, we further investigated which domain is responsible for the
association with eRF1. A FLAG-tagged full-length GSPT2 (amino acids
1-632) and its amino-terminal deletion mutant (amino acids 138-632)
were produced in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody. As shown in Fig. 1, a 54-kDa eRF1 protein of COS-7 cells
was observed in both of the two precipitated fractions, suggesting that
the amino-terminal region of GSPT2 is not involved in the binding to
eRF1. Essentially the same results were obtained if GSPT1 was used
instead of GSPT2 (data not shown).

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Fig. 1.
Association of amino-terminal deleted GSPT2
with eRF1. Expression vectors for FLAG-tagged full-length GSPT2
(amino acids 1-632; lane 1) and its amino-terminal deletion
mutant (amino acids 136-632; l ne 2) or the vector alone
(lane 3) were transfected into COS-7 cells, and the cell
extracts were immunoprecipitated with an anti-FLAG monoclonal antibody
(9). The precipitated proteins were subjected to immunoblot analysis
with anti-GSPT1 (A) and anti-eRF1 (B)
antibodies.
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Search for Other GSPT-binding Proteins: Association with
Poly(A)-binding Proteins via the Amino-terminal Domain of GSPT--
To
investigate possible function of the unique amino-terminal region of
GSPT and other molecules interacting with the GSPT family, we first
screened a human T cell lymphoma cDNA library with GSPT1 as a bait
in yeast two-hybrid assay system. The initial screening (6.5 × 106 independent clones) resulted in the identification of
10 positive clones. Sequencing analyses revealed that all clones
encoded PABP I (12). Positive interaction with PABP was also observed
with GSPT2 more effectively under the present conditions (data not shown). As shown in Fig. 2A,
PABP I contains four RNA-binding domains in tandem repeat at its
amino-terminal side; these domains bind to the 3'-poly(A) tail of
mRNAs probably for their stabilization and/or translocation from
nuclei to cytoplasm (for review see Refs. 13 and 14). The
carboxyl-terminal domain of PABP is suggested to contribute in
multiple, regularly spaced organization of the RNA-binding protein on
the poly(A) tail (15).

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Fig. 2.
Schematic representation of the primary
structure of mouse GSPTs and PABP I and analysis of the interaction
between GSPT2 and PABP in the yeast two-hybrid system.
A, the GSPT family consists of two regions characterized as
an amino-terminal nonhomologous region (approximately 200 amino acids)
and a conserved EF1 -like domain (428 amino acids). The EF1 -like
domain contains four GTP-binding motifs (G1-G4). A Glu-rich
(E-rich) region is present between the amino-terminal region
and EF1 -like domain, and a Gly repeat structure (GGGG) is only
present in GSPT1. PABP I consists of two regions, four RNA-binding
domains (1-4) in tandem repeat at the amino-terminal side and the
carboxyl-terminal portion. B, a series of the amino- and
carboxyl-terminal deletion mutants of mouse GSPT2 and human PABP I were
inserted into pGBT9 and pGAD424 vectors, respectively, and transformed
into SFY526 cells. -Galactosidase activity of the cell lysate was
measured as described under "Experimental Procedures." Values shown
are the means ± S.D. of triplicate determinations.
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For further characterization of the interaction between GSPT and PABP,
the varying lengths of GSPT2 and PABP were cloned downstream of the
GAL4-binding (pGBT9 vector) and activation (pGAD424 vector) domains,
respectively, and the resulting plasmids were transformed into the
S. cerevisiae SFY526 cells. As shown in Fig. 2B,
we observed strong
-galactosidase activity with the full-length
GSPT2 (amino acids 1-632) and PABP in the yeast two-hybrid assay. When
135 amino acids were deleted from the amino terminus of GSPT2 (amino acids 136-632), the interaction was abolished. In sharp contrast,
-galactosidase activity was markedly enhanced if the
carboxyl-terminal EF1
-like domain was deleted from GSPT2 (amino
acids 1-204).
Because PABP has the two-domain structure, we further analyzed which
domain is important for the binding to GSPT2. The interaction with
GSPT2 appeared to be mediated through the carboxyl-terminal side (amino
acids 369-633) of PABP rather than its amino-terminal RNA-binding
domain (amino acids 1-368). Such difference observed between the two
regions of PABP was also ascertained if the amino-terminal region of
GSPT2 (amino acids 1-204) was used instead of the full-length GSPT2
(amino acids 1-632). Thus, the interaction appeared to be mediated
through the carboxyl-terminal domain of PABP and the amino-terminal
region of GSPT.
Direct Interaction between GSPT and Poly(A)-binding
Protein--
The direct interaction between GSPT and PABP was further
confirmed by an in vitro binding assay with recombinant
proteins. E. coli cell lysates containing a constant amount
of GSPT1 were incubated with increasing amounts of purified GST-fused
PABP or GST alone, and glutathione-Sepharose resin was added to the
reaction mixture. Proteins bound to the resin were separated on a
SDS-polyacrylamide gel and immunoblotted with anti-GSPT1, anti-PABP,
and anti-GST antibodies. As shown in Fig.
3, there were increasing amounts of
90-kDa GSPT1 in the resin when the increasing amounts of GST-PABP were
added to the reaction mixture (lanes 2-4). Such interaction was not observed upon incubation with GST (lanes 5-7).
Essentially the same results were obtained if GSPT2 was used instead of
GSPT1 (data not shown).

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Fig. 3.
In vitro assay for the interaction
between GSPT1 and PABP. Crude E. coli extract
containing approximately 10 µg of GSPT1 was incubated with the
indicated amounts of purified GST-fused PABP or GST in TEDN, and then
glutathione-Sepharose beads were added to the mixture. Proteins bound
to the beads were separated by SDS-polyacrylamide gel electrophoresis
(12.5% acrylamide) and immunoblotted with anti-GSPT1 (A),
anti-PABP (B), and anti-GST (C) antibodies.
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Inhibition of Multimerization of PABP with Poly(A) by the
Amino-terminal Domain of GSPT--
We next investigated the functional
significance of the interaction between GSPT and PABP. Previous studies
indicated that the carboxyl-terminal domain of PABP confers
multimerization activity, which can be measured by gel mobility shift
assay (15). As shown in Fig. 4,
5'-32P-labeled, synthetic poly(A) RNA bound to PABP,
resulting in the formation of the retarded band. There was a
progressive reduction of the complex mobility indicative of more than
one copy of PABP on the labeled poly(A) as the amount of the
RNA-binding protein was increased (lanes 2-5).
However, in the presence of the amino-terminal domain of GSPT
(lanes 6-10), a discrete transition rather than a gradual reduction of the complex mobility was observed. It thus appeared that only one complex forms at saturating concentrations, although its mobility was slower than that of a single complex of
poly(A)-PABP probably due to the association of GSPT amino-terminal domain (compare lane 8 with lane 2). These
results indicate that PABP associated with GSPT in its carboxyl
terminus could no longer interact with another PABP to form a multiple,
regularly spaced complex on the poly(A) tail of mRNAs.

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Fig. 4.
Effect of amino-terminal region of GSPT2 on
interaction between PABP and poly(A) mRNA. The indicated
amounts of purified PABP (up to 10 pmol) was incubated with GST-fused
amino-terminal domain (amino acids 1-204) of GSPT2 or GST alone (40 pmol) in TEDN. The mixture was further incubated with 0.02 pmol
(23-mers) of 32P-labeled poly(A) RNA and subjected to gel
shift assay as described under "Experimental Procedures."
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Possible Functions of GSPT/eRF3 in Eukaryotic Translation
System--
The precise mechanism by which eRF1 and GSPT/eRF3 promote
translation termination when the A site of the ribosome is occupied by
a termination codon has not been fully elucidated. As shown in Fig.
5, one suggested model would be
comparable with the elongation system; eRF1 may structurally mimic the
stem of an aminoacyl-tRNA, whereas GSPT/eRF3 may mimic the function of
EF1
, which carries GTP-dependently the aminoacyl-tRNA to
the A site. This model may be consistent with the present finding that
the association with eRF1 required only the carboxyl-terminal domain of
GSPT/eRF3, which covers almost the full length of EF1
(Fig. 1).
Thus, the extra amino-terminal region (approximately 200 amino acid
residues) of GSPT/eRF3 appeared to be responsible for other functions.
In the present report, we identified that one of the possible functions is to associate with the carboxyl-terminal domain of PABP (Figs. 2 and
3). Furthermore, the amino-terminal domain of GSPT/eRF3 could inhibit
the formation of multimeric PABP-PABP interactions on poly(A) RNA (Fig.
4).

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Fig. 5.
A proposed model for the functions of
eRF3/GSPT in eukaryotic protein synthesis. When the A site of
ribosome is occupied by a stop codon, eRF3/GSPT carries eRF1 for the
translation termination via its carboxyl-terminal EF1 -like domain.
eRF3/GSPT also associates with PABP via its amino-terminal region for
the destabilization of mRNA and/or the initiation of new
translation cycle. For details, see "Possible Functions of
GSPT/eRF3 in Eukaryotic Translation System."
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These results led us to speculate that the extra region of
amino-terminal GSPT/eRF3 may consequently destabilize the association of PABP with 3'-poly(A) tail, leading to the degradation by
ribonuclease(s) of the mRNA recruited for the translation. In other
words, a signal from each translation termination cycle in the
ribosomal A site would be transferred to the 3'-poly(A) tail of
mRNA as a degradation signal via the interaction between GSPT/eRF3
and PABP. This idea may be supported by the findings that repetitive
translation shortens the 3'-poly(A) tail of mRNA and that PABP can
extend the lifetime of mRNA (13, 14, 16, 17). In this relation,
Czaplinski et al. (18) have quite recently reported another
similar function of eRF1/eRF3. They demonstrated that the product of
the UPF1 gene, which is a RNA-dependent ATPase
containing RNA helicase activity, interacts with the releasing factors
in a nonsense mutation-mediated mRNA decay pathway. The interaction
of UPF1 gene product with eRF1/eRF3 appears to enhance the
termination and degrade the aberrant mRNAs.
Moreover, it has recently been reported that the poly(A)-binding
domains of PABP can associate with the translation initiation factor
eIF4G, which binds to the 5'-cap structure of mRNA through eIF4E to
promote the translation initiation (for review see Refs. 19 and 20).
Thus, the signal from the translation termination cycle might also
regulate the new initiation step via the eRF3/PABP/eIF4G signaling
cascade. It will be very interesting to determine how the GDP-GTP
exchange reaction and/or the GTPase cycle are involved in the
multi-functions of GSPT/eRF3. In any event, the results presented here
have identified GSPT/eRF3 as a regulator of PABP interacting with the
3'-poly(A) tail of mRNAs, suggesting that it may also play an
important role in the degradation of mRNAs and/or the regulation of
translation efficiency mediated through the initiation factor(s).
Clearly, further experimentation is required to test this fundamental hypothesis.