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J Biol Chem, Vol. 274, Issue 50, 35991-35998, December 10, 1999
From the Department of Biochemistry and Molecular Biology,
Louisiana State University Medical Center,
Shreveport, Louisiana 71130-3932
Ribonucleotide reductase synthesizes dNDPs, a
specific and limiting step in DNA synthesis, and can participate in
neoplastic transformation when overexpressed. The small subunit
(ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a
subtraction library from eukaryotic initiation factor 4E
(eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)). CHO-4E
cells have 20-40-fold elevated RNR2 protein, reflecting an increased
distribution of RNR2 mRNA to the heavy polysomes.
CHO-4E cells display an altered cell cycle with shortened S phase,
similar to cells selected for RNR2 overexpression with
hydroxyurea. The function of ribonucleotide reductase as a checkpoint
component of S progression was studied in yeast in which elevated eIF4E
rescued S-arrested rnr2-68ts cells, by
increasing recruitment of its mRNA to polysomes. Crosses between
rnr2-68ts and mutant eIF4E
(cdc33-1ts) engendered conditional synthetic
lethality, with extreme sensitivity to hydroxyurea and the microtubule
depolymerizing agent, benomyl. The double mutant (cdc33-1
rnr2-68) also identified a unique terminal phenotype, arrested
with small bud and a randomly distributed single nucleus, which is
distinct from those of both parental single mutants. This phenotype
defines eIF4E and RNR2 as determinants in an important cell cycle
checkpoint, in early/mid-S phase. These results also provide a link
between protein and DNA synthesis and provide an explanation for cell
cycle alterations induced by elevated eIF4E.
The translation initiation factor eukaryotic initiation factor 4E
(eIF4E)1 recognizes the
7-methylguanosine-containing cap of mRNA, in the first step of
mRNA recruitment for translation (1). It is assumed to be the least
abundant of the initiation factors and is rate-limiting for translation
of some mRNAs (2, 3). eIF4E functions as the cap-binding subunit of
eIF4F, the complex that unwinds secondary structure at the 5'
untranslated region (5'-UTR) of mRNAs in an ATP-dependent reaction. This function is critical during
scanning for exposing and locating the translation start site (4-6).
In Saccharomyces cerevisiae, eIF4E was cloned as a cell
division cycle gene (CDC33), essential for cell viability
(7, 8). CDC33 is involved in the G1-S transition
of the cell cycle. The temperature sensitive mutant strain,
cdc33-1, arrests in G1 (before Start) with
small, unbudded cells at the restrictive temperature.
Overexpression of eIF4E causes deregulated cell growth and malignant
transformation of rodent and human cells (9-11). Hence, the mechanism
of transformation by eIF4E presents an important and challenging
biological phenomenon. Furthermore, eIF4E is elevated in common human
neoplasms, such as carcinomas of the breast and the head and neck (12,
13). One explanation is that the translation of certain proto-oncogene
or growth factor transcripts, which normally are translationally
repressed, becomes preferentially increased by an excess eIF4E.
Accordingly, a few of these have been identified, e.g. c-Myc
and ornithine decarboxylase (ODC) (reviewed in Refs. 14 and 15).
However, a systematic identification of the transcripts of which the
translation is increased by the excess eIF4E would represent a major
step forward in understanding the role of dysregulation of protein
synthesis in cancer etiology. We have addressed this task with the
construction of a subtraction cDNA library from the heavy polysomes
of CHO cells overexpressing eIF4E (CHO-4E). From an initial screening,
we identified an abundantly represented product corresponding to the
small subunit of ribonucleotide reductase (RNR2).
Ribonucleotide reductase (RNR) catalyzes the conversion of
ribonucleoside diphosphates (NDPs) into deoxyribonucleotides (dNDPs), one of the most specific and rate-limiting steps in DNA synthesis. The
RNR holoenzyme is a tetramer composed of two large subunits (RNR1) and
two small subunits (RNR2). RNR2 maintains a stabilized tyrosyl radical
essential for the reduction of ribose. RNR1 harbors redox-active
cysteines and allosteric sites to maintain balanced pools of
nucleotides. Given the huge energetic investment of generating the
necessarily large pool of dNTPs for DNA replication and repair, it is
not surprising that RNR expression is carefully regulated and
restricted to the late G1 and S (and G2) phases
of the cell cycle or to conditions of DNA damage (16-19). In mammals,
only the expression of RNR2 is regulated, reportedly at the
transcriptional level (20). In contrast, RNR1 is
constitutively expressed throughout the cycle. In S. cerevisiae, RNR1 is cell cycle-regulated at the transcriptional level (18). The level of dNTPs can be rate-limiting for
DNA replication. This is evidenced from the fact that temperature sensitive mutants of RNR in yeast traverse the S phase very
slowly and arrest in S phase at the restrictive temperature with large budded cells (19, 21). Agents that inhibit dNTPs synthesis, such as
methotrexate, are used to inhibit replication of cancer cells;
conversely, elevated RNR may hasten the cell division. Accordingly,
RNR2 was recently recognized as a powerful transforming agent in cooperation with a variety of oncogenes in mammalian cell
lines (22, 23). RNR2 was also identified as an overexpressed product in a panel of breast carcinomas (24).
We propose that increased expression of RNR2 in cells with
elevated eIF4E represents an important avenue by which eIF4E can induce
malignant transformation. Moreover, the evidence herein for
translational regulation of RNR constitutes perhaps the most direct link between a central player in protein synthesis (eIF4E) and a
key enzyme in DNA synthesis.
Cell Lines and Tissue Culture--
Chinese hamster ovary cells
stably transfected with BK or BK-4E episomal vectors were cultured as
described previously (10).
DNA Synthesis and Cell Cycle Synchronization--
For
synchronization experiments, CHO-BK and CHO-4E cells were arrested in
G0/G1 or in early G1 with some
modifications of the published procedure (25). In brief, aliquots of
106 cells grown on 60-mm plates were arrested at
G1/S with 5 µg/ml aphidicolin (Sigma) for 18 h,
released from the block with three washes of fresh medium, and allowed
to grow for 12 h. Finally, the cultures were serum-starved for
12 h. This treatment resulted in greater than 80% of the cells
arrested in G0/G1 or early G1 phase
of the cell cycle. DNA synthesis was measured by incorporation of
[6-3H]thymidine (15 Ci/mmol; NEN Life Science Products)
added directly to the medium (15 µCi/ml). At specific time points,
the label was removed, and the cells were washed twice with
phosphate-buffered saline and lysed with 1 ml of 2% SDS. 10%
Trichloroacetic acid-precipitable material was collected on GF/A
filters (Millipore, Bedford, MA) and counted. The cpm were normalized
for absorbance at 260 nm of total cell lysate.
Polysomal RNA Isolation and Analysis--
CHO-BK and CHO-4E
cells were briefly treated with cycloheximide at a final concentration
of 150 µg/ml (Sigma) to freeze the ribosomes and harvested with 2.5 mM EDTA in phosphate-buffered saline. Polysomal RNA was
obtained after collection of gradient fractions, as described (26).
Alternatively, the samples were centrifuged for 4 h to pellet
heavy polysomal RNA (P100), with a sedimentation value of Construction of the Subtraction cDNA Library--
The P100
fraction was dissolved in 50 mM Tris/0.5% SDS and digested
by proteinase-K (1 mg/ml). Poly(A)-containing RNA was isolated by
chromatography on oligo(dT)-cellulose (Amersham Pharmacia Biotech, type
7). Construction of the cDNA library from the polysomal RNA
involved the following steps. 1) The first-strand cDNA was synthesized using SuperScript II (Life Technologies, Inc.) with the
primer (PA-S): 5' TAG TCG ACT TTT TTT TTT TTT TTT 3'. Template RNA was
then removed by incubation in alkali. 2) The CHO-4E single-stranded cDNA was depleted (subtracted) of common transcripts by
hybridization with a 2-fold excess of polysomal mRNA from control
cells (P100 of CHO-BK). The hybridization was carried out in 0.2% SDS,
2 mM EDTA, 600 mM NaCl, 20 mM
Tris-HCl (pH 7.5), at 68 °C for 24 h. The resulting hybrid
duplexes were removed by adsorption to glass milk (Bio 101, La Jolla, CA) in 0.5× phosphate-buffered saline. The remaining
single-stranded cDNA (unbound) was ETOH-precipitated, and dC-tailed
with terminal-dNTP-transferase. 3) A 5'-end primer (PG-N: 5' TAG CGG
CCG CGG GXX GGG XXG GGX XG 3') and Klenow polymerase was used for
second-strand synthesis, and the same set of primers was then used in
20 cycles of polymerase chain reaction to slightly amplify the
cDNA. (The polymerase chain reaction profile was 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 5 min). 4) Cloning of the
cDNA library into pBluescript KS Sequencing of Clones and Computer Analysis--
cDNA inserts
from the clones were sequenced on both strands using
Sequenase II (USB), and derived sequences were analyzed by
Blast (GenBankTM, NCBI, National Institutes of Health).
Slot Blots and Northern Blots--
Aliquots from sucrose
gradient fractions were filtered through BioBlot-NC (Costar) using a
Minifold II Slot Blot apparatus (Schleicher & Schuell) and fixed onto
the membrane with 10% formaldehyde for 3 min. The membrane was
prehybridized with 7% SDS, 0.5 M
Na2HPO4 (pH 7.4), 10 mM EDTA at
45 °C for 3 h and hybridized overnight at 45 °C in the same
buffer containing the appropriate riboprobes labeled with
[ Western Analysis of Cell Lysates--
20 or 30 µg of protein
of each sample was separated on a 10% SDS-polyacrylamide gel
electrophoresis gel. The proteins were transferred to Immobilon-P
membrane (Millipore). Anti-lactic dehydrogenase serum was from Sigma.
The anti-tubulin/anti-RNR2 rat antibody (YL1/2, Serotec, Oxford, United
Kingdom) was biotinylated and then purified on protein G-Sepharose
(Sigma). After primary antibody reaction, the blots were reacted with
peroxidase-coupled ExtraAvidin (Sigma) (1:1000 dilution) for 1 h.
Finally, the membranes were washed three times and developed by
chemiluminescence (ECL, Amersham Pharmacia Biotech).
Yeast Strains and Media--
Two S. cerevisiae ts
mutant strains, rnr1-240 (MATa ade2-1, his3-11, 15, leu2-3, 112, trp1-1, ura3-1 rnr1-240) and rnr2-68 (MAT Overexpression of eIF4E in Yeast--
The eIF4E expression
plasmid, YCpIF15-CDC33, containing the eIF4E ORF (CDC33)
under the control of a GAL1 promoter, was constructed by
cloning the 0.75-kb SpeI fragment in frame with an
hemagglutinin tag. Cells carrying either YCpIF15 or YCpIF15-CDC33 were
grown to an early log phase in liquid culture medium lacking tryptophan (for selection of the plasmid) and containing 2% raffinose. 2% galactose was added to these cultures as indicated to induce
overexpression of eIF4E. Doubling times (generation time) were
determined by direct counting and spectrophotometry.
Measurement of HU and Benomyl Sensitivity--
Mid-log phase
cultures of yeast were sonicated and counted. 10-Fold dilutions from
105 to 10 cells were spotted on YPAD or YPGal plates
containing HU (0.1-200 mM) or benomyl (1-40 µg/ml).
Plates were incubated at room temperature (23 °C) for 3-5 days. For
each genotype, at least two sets of experiments were done.
Fluorescence Microscopy and Fluorescence-activated Cell Sorting
Analysis--
Exponentially growing cultures in YPAD liquid medium at
22 °C was shifted to 30 °C. After the cells arrested, cellular
DNA was visualized by staining with 4',6'-diamidino-2-phenylindole as
described (30).Cells were prepared for fluorescence-assisted cell
sorting by staining with propidium iodide as described (31).
Excess eIF4E Elicits a Selective Redistribution of mRNA Classes
in the Heavy Polysomal Fractions--
We previously reported that
overexpression of eIF4E in CHO (or continuous rat embryo fibroblasts)
cells resulted in a transformed phenotype, which included loss of
contact inhibition, shortening of generation time, and growth in soft
agar (11). The level of eIF4E was increased 7-fold (uninduced), and
20-fold following induction of the promoter with
tetrachlorodibenzodioxin, compared with control cells. Consistent with
this elevation in eIF4E, the overall rate of protein synthesis was
increased by 30 and 50% without and with tetrachlorodibenzodioxin,
respectively. However, SDS-polyacrylamide gel electrophoresis
autoradiography of the newly synthesized proteins revealed that whereas
the synthesis of most proteins was only modestly increased, the
synthesis of several polypeptides was greatly enhanced (11). This
result is in agreement with a model treatment of mRNA competition
for translation initiation, which predicts that the less competitive or
weak mRNAs will benefit the most from the excess eIF4E
(Ref. 32; reviewed in Ref. 15). In an attempt to identify some of these
products, we initially carried out Western blots for several important
regulators of cell growth. One identified product is basic fibroblast
growth factor (FGF-2), which was increased 40-50-fold in CHO-4E
compared with the control, CHO-BK (26). Fig.
1A shows that this increase is
fully attributable to increased translation initiation of FGF-2
mRNA, as shown by enhanced utilization and recruitment to the heavy
polysomes. The weighted average increase in FGF-2 signal in the large
polysomes of CHO-4E is 4-fold; the average load in this region of the
gradient is 10 ribosomes per transcript. Thus, a 4-fold increase in
these fractions should result in (the observed) 40-fold increase in
FGF-2 synthesis per unit time. Note that the polysomal distribution of
glyceraldehyde-3-phosphate dehydrogenase, a typical strong
(or competitive) mRNA, is not different in CHO-BK versus
CHO-4E cells (Fig. 1A). This result is consistent with the
model of competition for mRNA recruitment to the ribosomes
mentioned above and also forms the theoretical basis for generation of
an enriched cDNA library.
Briefly, poly(A)-containing RNA from the heavy polysomes of CHO-4E
cells provided the source for synthesis of the first-strand of
cDNA; after hybridization with an excess of polysomal mRNA from
control cells (CHO-BK), common transcripts were physically removed (see
under "Experimental Procedures"). To simplify the process, instead
of collecting multiple gradient fractions, we extended the
centrifugation time from 2 (Fig. 1A) to 4 h to sediment particles corresponding to RNR2 Is a Major Product Identified in a Subtraction Library from
the Heavy Polysomes of CHO-4E--
The cDNA library described
above was initially tested for enrichment by microarray display and
quantitative polymerase chain reaction, using primers for typical
strong mRNAs (e.g. glyceraldehyde-3-phosphate dehydrogenase) versus weak mRNAs (e.g. FGF-2
and vascular endothelial growth factor) previously known to be
preferentially recruited by the excess eIF4E (26, 34). Following these
preliminary tests (data not shown), we analyzed the cDNA inserts by
restriction digests and partial sequencing. Among the first identified
cDNA inserts, there were several independent clones corresponding
to the RNR2 mRNA of golden hamster (Mus
auratus; 97% homology). To confirm that RNR2 is a
translationally regulated product in cells transformed with eIF4E, we
probed the Northern blot in Fig. 1B with a partially
sequenced clone, corresponding to nucleotides 2212-3064 of
RNR2 of M. auratus. The Northern blot revealed a transcript of the expected size (~3 kb) (35), and it demonstrated a
reciprocal redistribution of the RNR2 mRNA in the P100
and S100 fractions (Fig. 2A).
There was a 6-fold increase in RNR2 mRNA in the P100 of
CHO-4E compared with CHO-BK, as measured by densitometric analysis and
by reprobing the slot-blot in Fig. 1A (data not shown). The
total amount of RNR2 mRNA was similar in the two cell
lines (Fig. 2A, right panel). This result proves that
RNR2 mRNA is engaged for translation initiation far more
efficiently in CHO-4E cells and that it may fall into the category of
translationally repressed transcripts.
To confirm that RNR2 is overexpressed in CHO-4E, we
determined the level of RNR2 protein by Western blot. The blot was
probed with the antibody YL1/2, raised against the yeast The Duration of S Phase Is Shortened in CHO-4E Cells--
CHO-4E
cells divide more rapidly than control CHO-BK and can attain a higher
saturation density (11). We thus determined the length of the cell
cycle in CHO-BK and CHO-4E cells. To do this, we obtained a culture
with 80% of cells arrested at G0/G1 (see under
"Experimental Procedures"). Mitogenic stimulation, by the addition
of serum-containing medium, resulted in a rapid entry into the cell
cycle, with DNA synthesis beginning 6 h later for both CHO-BK and
CHO-4E cells. The average generation time of CHO-4E was shorter (17.5 h) than that of CHO-BK (21 h). Synthesis of DNA was completed by
13 h post-serum stimulation in CHO-4E cells, whereas completion
did not occur until 17 h in CHO-BK (Fig. 3). This shortening of the S phase
constitutes the bulk of the reduction in generation time of CHO-4E
cells. Similarly, cells selected for elevated expression of
RNR2 by progressive selection in hydroxyurea (CHO-HU in Fig.
3) (35) also displayed a shorter S phase, completed in 14 h. These
cells have a 5-fold increase in RNR2 (data not shown). It seems
plausible that the excess RNR2, the limiting component of RNR in
mammals, could speed up the replication period by making a larger pool
of dNTPs available to the DNA replication machinery. However, these
data cannot exclude the possibility of activation of an additional
pathway that may facilitate the DNA replication process.
CHO-4E Cells Are Naturally Resistant to Hydroxyurea--
HU
specifically inactivates RNR by scavenging the tyrosyl radical at the
catalytic site of RNR2. Overexpression of RNR2, but not of
RNR1, was shown to increase cell resistance to HU (16, 35).
We thus tested whether CHO-4E cells were more tolerant to growth
inhibition by HU. The cells were grown in 24-well dishes (in
duplicates) and treated for 8 h with increasing concentrations of
HU. The cells were then incubated for 17 h in presence of 10 µCi
of [3H]thymidine to measure the inhibition of DNA
synthesis by HU (see under "Experimental Procedures"). The
calculated LD50 for CHO-BK was near 0.1 mM,
whereas it was 1 mM for CHO-4E (data not shown). This
confirmed that CHO-4E cells are more resistant to HU, consistent with
their elevated expression of RNR2.
Overexpression of eIF4E Restores Growth to a rnr2-68 Yeast
Strain--
The alteration in cell cycle caused by eIF4E prompted us
to determine whether elevated eIF4E generally increases RNR
expression in other systems, with corresponding effects on the cell
cycle. Thus, we took advantage of a S. cerevisiae strain,
rnr2-68, harboring a ts mutation in
RNR2. At the restrictive temperature (34 °C) this strain
stops growing and arrests in S phase with abnormally large buds. At the
permissive temperature (23 °C), the cells are viable with the
majority traversing the S phase slowly. This strain was transformed
with a vector expressing hemagglutinin-tagged eIF4E (CDC33)
under the control of a GAL1 promoter
(YCpIF15-CDC33), or with a control vector lacking
CDC33. Functional activity of this hemagglutinin-tagged
eIF4E was confirmed in vivo by its ability to complement
cdc33-1ts mutant at 37 °C (data not shown).
Trp+ YCpIF15-CDC33 transformants were streaked onto
synthetic medium containing 2% galactose, to induce the expression of
eIF4E. As shown in Fig. 4A,
overexpression of eIF4E restored growth to the rnr2-68
strain at nonpermissive temperature. By contrast, rnr2-68
containing YCpIF15 (control vector) was unable to grow at 34 °C.
This suppression of heat sensitivity of rnr2-68 may be due
to either an increase in the expression of rnr2-68 allele
due to excess eIF4E or activation of an alternate pathway that allows
cells to bypass the mutation.
At permissive temperature, the doubling time of the mutant,
rnr2-68, is 8 h. The doubling time was reduced by
approximately 50% when overexpression of eIF4E was induced with
galactose. Thus, rnr2-68 grows better even at permissive
temperature when eIF4E is overexpressed.
The Double Mutant, cdc33-1 rnr2-68, Shows Conditional Synthetic
Lethality--
To further elucidate the relation between eIF4E and
RNR2 expression, we constructed a cdc33-1
rnr2-68 double mutant by crossing the single mutants (see under
"Experimental Procedures"). After tetrad dissection, all double
mutants obtained from tetratype and nonparental ditype gave rise to
very small colonies compared with the single mutants (Fig.
4B). At permissive temperature (20 °C), double mutant
cells showed a very slow growth phenotype with extreme sensitivity to
HU (unable to grow on plates containing 1 mM of HU) and to
the microtubule depolymerizing drug benomyl (unable to grow on plates
containing more than 5 µg/ml). The wild-type parental strain (CRY1)
is resistant up to a concentration of 200 mM HU and 15 µg/ml benomyl. The single mutants, cdc33-1 and
rnr2-68, can grow up to 34 °C; the double mutant showed
conditional synthetic lethality at 28 °C in rich medium (Fig.
4C). Fig. 4D shows that the double mutant arrests
in S phase at 28 °C as singly budded (Nomarski),
uninucleated cells (Dapi). Note that this is different from
cdc33-1 cells, which arrest unbudded. DNA content in these arrested cells was shown to be 1 N by propidium iodide
staining and flow cytometry (data not shown). Together, these results
indicate that CDC33 indeed exerts its effects on the cell
cycle by modulating the expression of RNR2 as one of its
downstream effectors. However, it is likely that some other cell
components, in addition to RNR, are also affected by the
reduction of functional eIF4E. In the double mutant, the smaller bud
size and random distribution of the undivided nucleus in either mother
or daughter cells are indicative of this (Fig. 4D). In
contrast, the typical S phase-arrested rnr2-68 shows
abnormally large-budded cells with nucleus being either in the mother
cell or near the bud neck, but never in the daughter cell.
eIF4E Regulates the Expression of RNR2 at the Translational Level
in Yeast--
The evidence provided above suggested that the
expression of RNR2 is regulated by eIF4E in yeast as well.
In order to see whether this control by eIF4E is at the transcriptional
and/or translational level, we tested the in vivo effects of
overexpression of eIF4E on the message level of RNR2 and on
the recruitment of RNR2 mRNA onto the polysomes. The
polysomal fractions were obtained from an exponentially growing culture
of a wild-type strain containing either control vector or the eIF4E
overexpressing vector (YCpIF15-CDC33), grown in minimal
medium without Trp containing 2% raffinose + 2% galactose. The
Northern blot revealed a redistribution of the RNR2 mRNA
in the P100 and S100 fractions in the CDC33-overexpressing strain compared with the control. Fig.
5A shows that there is a
6-fold increase in RNR2 mRNA in the P100 fraction of
eIF4E-overexpressing strain compared with that of CRY1-control, as
measured by densitometric analysis of this and another slot-blot (data
not shown). However, the total amount of RNR2 mRNA did
not change in the two strains (data not shown). This indicates that
there is an increase in the translation initiation rate of
RNR2 in eIF4E-overexpressing cells.
We also examined the effects of depletion of functional eIF4E
(CDC33) on the expression of RNR2. The congenic
cdc33-1 ts strain that arrests in G1 at
34 °C or above (8) was tested in a temperature shift experiment.
When an exponential culture of this strain grown in YPAD was shifted
from 23° to 37 °C, RNR2 protein was drastically reduced within
1-2 h of incubation, whereas the level of eIF4E is a powerful oncogene/growth activator when overexpressed
in model cell lines (9-11). Furthermore, eIF4E is elevated 3-30-fold
in breast carcinomas but not in benign lesions, indicating that its
overexpression may mark a critical transition in the genesis of breast
cancer (12, 38). We have proposed that eIF4E causes some of its effects
by specifically increasing the translation of certain growth factors
and important regulators of cell growth (reviewed in Ref. 15). The low
abundance of eIF4E creates a situation of competition among different
mRNA species, such that weak mRNAs are outcompeted
for binding to ribosomes by the strong mRNAs (39). An
analysis of sequence data from 700 vertebrate mRNAs has shown that
more than 90% contain 5'-UTRs that are less than 200 nucleotides long
and devoid of upstream AUGs, which is characteristic of
strong mRNAs (40, 41). In contrast, weak mRNAs contain long G-C-rich 5'-UTRs, with potential for forming stable secondary structure, and/or upstream AUGs (42, 43). An excess of
eIF4E results in a selective advantage for the translation of
weak mRNAs, for example FGF-2 and ODC.
In an attempt to carry out a systematic analysis of the proteins that
are strongly increased by eIF4E, we made a subtraction cDNA library
from the polysomes of CHO-4E cells. Several independent clones (4% of
the total pool of transformants) were identified as RNR2,
and its identity as a major translationally up-regulated transcript was
subsequently confirmed by Northern and Western blots. The expression of
RNR2 was not altered transcriptionally, because its mRNA
level was the same in CHO-4E and CHO-BK cells; only its distribution in
S100 and P100 fractions was altered.
The increased expression of RNR2 (and consequently of RNR)
provides one possible explanation for the observation that neither the
duration of G1 nor that of G2+M was altered in
CHO-4E (data not shown). This seemed in stark contrast with the fact
that the generation time was reduced by 20% in CHO-4E
versus control cells. This apparent paradox was resolved
with the finding that the S phase transit period was shortened (by 3-4
h) in CHO-4E, demonstrating that these cells can complete DNA
replication faster than normal. At this point, we do not know precisely
how an increase in RNR is translated into a corresponding increase in
the rate of DNA replication in CHO-4E. However, cells selected for
elevated expression of RNR2 by progressive selection in
hydroxyurea (CHO-HU) also showed a shorter S phase. Because synthesis
of deoxyribonucleotides is rate-limiting for DNA synthesis in most
organisms, an excess of RNR2 may allow for a faster replication of the
genome, especially considering that replication in eukaryotes proceeds
simultaneously from multiple origins.
Translational regulation of RNR2 is well known for clam and
sea urchin eggs, in which fertilization triggers its synthesis as one
of the most abundant products of maternal mRNAs (36). Translational
regulation of RNR has not been reported in other systems, except for
the finding of an instability determinant in the 3'-UTR of
RNR2 mRNA (44). However, this is the first report of a
specific translational enhancement by eIF4E, classifying RNR2 as a weak mRNA and suggesting modulation
by its 5'-UTR. The function of eIF4E is to nucleate the assembly of
preinitiation complexes at the mRNA 5'-UTR, although the 3'-UTR can
also participate in this process (45). Our data indicate that
translation of RNR2 is specifically facilitated by excess
eIF4E. This was demonstrated by an analysis of polysomal profile of
RNR2 mRNA in cells overexpressing eIF4E. Interestingly,
suc22+, the RNR2 homolog of S. pombe,
is normally expressed as a 1.5-kb transcript, but upon treatment with
hydroxyurea a 1.9-kb transcript with an extended 5'-UTR is induced
(46), suggesting that it may be translationally regulated as well. The
same phenomenon was observed for the RNR4 gene, a
RNR2 homolog of S. cerevisiae (47, 48).
We also showed that inducible overexpression of eIF4E rescued viability
in a yeast strain harboring a ts mutation in the small subunit of RNR (rnr2-68) at the restrictive temperature.
Overexpression of eIF4E also compensated growth defects of the
rnr2-68 mutant as evidenced by a 50% reduction in
generation time. Our experiments do not rule out the possibility that
excess eIF4E may also result in an increase in RNR1 in this strain.
However, note that overexpression of RNR1 in the
rnr2-68 strain could not restore growth at
34 °C,2 demonstrating that
the intracellular RNR1 level is not limiting in this strain (see also
in Ref. 19). Previously, RNR1 was reported to be cell
cycle-regulated at the transcriptional level, fluctuating 15-30-fold
(18), whereas the RNR2 transcript showed no more than a
2-fold change during the cell cycle. It would then follow that
RNR2 must be translationally regulated during the cell
cycle, in order to be stoichiometric with RNR1. In this context, we
should recall that eIF4E activity fluctuates during the cell cycle
(25), and conversely, eIF4E was originally cloned as a ts
mutant controlling cell cycle progression in yeast (CDC33,
Ref. 8). Taken together with the results described here, it is likely
that transcriptional and translational regulation of the RNR
genes is an ancient, evolutionarily conserved mechanism. This is
further supported by the fact that the double mutant of
cdc33-1 and rnr2-68 could not grow at
temperatures above 28 °C, suggesting conditional synthetic
lethality. We also reported that the double mutant presents with a cell
cycle arrest that is different from either of the single mutants. The
fact that cdc33-1 rnr2-68 double mutant did not arrest in
early G1 obviates the possibility that the phenotype seen
in the double mutant was solely due to a direct effect of loss of
functional eIF4E. In contrast, the single mutant rnr2-68
strain arrests in the middle of S phase with large buds at restrictive
temperature, reflecting the fact that RNR first appears in late
G1 and reaches its peak in S phase (18, 19). The S phase
arrest in the double mutant has a different phenotype from that of
rnr2-68, as evidenced by their differences in cell
morphology (small buds), distribution pattern of nucleus, and
sensitivities to HU and benomyl. These argue in favor of the hypothesis
that the double mutant displays a phenotype that depends on both
cdc33-1 and rnr2-68 functions. The S phase
arrest in the double mutant can then occur by the following models.
cdc33-1 cells grow slowly and traverse G1 much slower than wild-type cells even at permissive temperature (8). The
decreased translation of the rnr2-68 mRNA (and
presumably some other G1 or G1 to S specific
mRNAs) caused by a defective cdc33-1 in the double
mutant may further slow down the cells in late G1. This may
result in an increase in the cell size that allows the cells to bypass
the size checkpoint in G1 and to proceed into S with a very
low level of active RNR. Plausibly, this results in the early S phase
arrest seen in this strain with small budded cells. The random
distribution of the unduplicated nucleus, either in mother or in
daughter, in this strain indicates that the coordination between
chromosome replication and segregation of nuclear material is affected
in these cells. This suggests that some cellular component(s) critical
for spindle formation or microtubule assembly and/or attachment of
chromosome to spindle pole may also be affected in this strain.
Supersensitivity of this strain to microtubule depolymerizing agent,
benomyl, also supports this notion. Alternatively, the
rnr2-68 in the double mutant, with a lengthened
S/G2 phase, may enable the cell to end up with a larger
size than usual after cytokinesis. Subsequent extension of the
pre-G1 period may also contribute to a bigger size of the
cells to begin with in the next cycle. Then, the cdc33-1
mutation slows down G1, which may result in a delay at or
around Start and a further increase in the cell size in G1
(size checkpoint). One can imagine that this could suppress the
pre-Start G1-arrest normally caused by cdc33-1 at restrictive temperature.
In retrospect, it is not surprising that synthesis of RNR, a key enzyme
in DNA replication, should be placed under translational control. In a
sense, the step at which synthesis of dNTPs commences is the most
direct indication that the cell is metabolically prepared to undertake
the expenses of replication. Such an endeavor cannot be limited to the
presence of favorable mitogenic signals from the environment, but it
also necessarily requires assessing of a replication-competent
enzymatic milieu, of which fully active protein synthesis is perhaps
the best indicator (49). Similarly, translational control of
RNR2 upon DNA damage would be very advantageous because of
the rapidity of such a response, which does not require de
novo gene expression. Very recently, it has been shown that in
addition to transcriptional control of RNR1, a
posttranslational component plays an important role in the regulation
of RNR activity. Sml1p inhibits dNTP synthesis by binding directly to
RNR1 subunit in yeast (50).
Exogenous expression of RNR2 has been recently recognized as
a powerful enhancer of transformation in cooperation with several oncogenes (22, 23). Interestingly, many large DNA tumor viruses (herpeviridiae and Epstein-Barr virus) encode their own genomic complement of RNR2 (and RNR1 in some cases)
rather than relying on the limiting cellular enzyme (51). The
RNR2 gene is also frequently amplified in drug-resistant
cancers, particularly following treatment with hydroxyurea or
MDL101731, another specific inhibitor of RNR2 (52, 53). Furthermore,
increased expression of RNR2 is recognized as an early
change in ductal carcinomas in situ (24) and in atypical
hyperplastic oral lesions (54). Coincidentally, these are the same type
of lesions that we have been studying as the earliest pathological
abnormalities in which overexpression of eIF4E becomes apparent (38,
55). Thus, we propose that, as the level of eIF4E increases during
malignant progression, there is a corresponding increase in
RNR2 translation and RNR assembly, leading to accelerated
cell replication and increased tolerance to DNA damaging agents. As
such, the identification of RNR2 as a major protein increased by excess
eIF4E adds a new important player to the limited spectrum of
translationally enhanced mRNAs that clearly play a key role in
eIF4E-induced cell transformation. The fact that elevated eIF4E, with
its corresponding effect on RNR2 expression, may naturally
protect cells from common chemotherapeutic drugs adds another reason to
design strategies to target eIF4E in cancer therapy.
We are indebted to M. Huang and S. J. Elledge (Baylor College of Medicine) for their generosity in providing
the yeast strains (rnr1-240, rnr2-68, and CRY1)
and the plasmids containing the yeast RNR1 and
RNR2. We are thankful to K. Matsumoto of Nagoya University
(Nagoya, Japan) for the plasmid YEp24CDC33 and the strain
cdc33-1. We thank D. Gross and K. Tatchell of this
department for the plasmids pGEM-ACT1 and YCpIF15,
respectively, and also for the helpful discussion. We thank D. Dempsey
for technical assistance in fluorescence-assisted cell sorting analysis.
*
This work was supported by National Science Foundation Grant
MCB9513756 and National Institutes of Health Grant CA69148.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.
§
These authors contributed equally to this paper.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.:
318-675-5668; Fax: 318-675-5180; E-mail: adeben@lsumc.edu.
2
M. R. Abid, Y. Li, C. Anthony, and A. De
Benedetti, unpublished observation.
The abbreviations used are:
eIF4E, eukaryotic
initiation factor 4E;
RNR, ribonucleotide reductase;
CHO, Chinese
hamster ovary;
FGF, fibroblast growth factor;
ODC, ornithine
decarboxylase;
kb, kilobase(s);
HU, hydroxyurea;
YPAD, yeast
extract-peptone-adenine-dextrose;
YPGal, yeast
extract-peptone-adenine-galactose;
UTR, untranslated region.
Translational Regulation of Ribonucleotide Reductase by
Eukaryotic Initiation Factor 4E Links Protein Synthesis to the Control
of DNA Replication*
§,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6
ribosomes. Yeast ribosomes were harvested from the exponentially
growing cell cultures by immediately cooling on ice and then by the
glass bead disruption method as described (27).
was as follows. Vector and
cDNA were digested with SalI and NotI,
ligated, and used to transform XL1-Blue supercompetent cells
(Stratagene, La Jolla, CA). The size of the inserts typically varied
between 300 and 7500 base pairs.
-32P]UTP (ICN). For isolation of RNA from the S100
(fewer than 5 ribosomes), the supernatant was mixed with an equal
volume of 3% CETAB (cetyl-trimethylammonium bromide, Sigma). RNA was
precipitated by centrifugation at 30,000 × g. The next
steps were similar for extraction of RNA from S100 and P100: the
precipitates were redissolved in 200 µl of 5 M
guanidine-HCl and extracted with phenol/chloroform. The RNA was
precipitated by ethanol, washed, dried, and redissolved in 200 µl of
DEPC-H2O. For Northern blots, the RNA was denatured with
glyoxal/Me2SO, separated on 1.3% agarose gels and
transferred to an Immobilon-Ny+ membrane (Millipore Co.), and
hybridized as above. For yeast slot-blot analysis, hybridization was
done by using a 32P-UTP labeled riboprobe of the yeast
RNR2. For Northern analysis, total yeast RNA was extracted
by using hot-phenol glass bead procedure as described (56). The
32P-labeled probes against RNR1 and
RNR2 mRNAs were synthesized by random priming of
oligonucleotides (Amersham Pharmacia Biotech).
ade2-1, his3-11, 15, leu2-3, 112, trp1-1, ura3-1
rnr2-68) were generously provided by S. J. Elledge (Baylor
College of Medicine, Houston, TX). The cdc33-1
(MAT his3 leu2 ura3 trp1) strain was back-crossed to the
wild-type, parental strain of the rnr mutants (CRY1) six
times to obtain a congenic cdc33-1 strain. During each cross, the presence of cdc33-1 allele was confirmed by 2:2
co-segregation of its G1 arrest and temperature sensitive
phenotype. The double mutant cdc33-1 rnr2-68 was obtained
by mating two congenic strains, rnr2-68 and
cdc33-1. The resultant diploid was subject to tetrad dissection (see Fig. 4B) after sporulation. The slowly
growing double mutants were confirmed by back-crossing with an isogenic wild-type haploid followed by tetrad dissection of the diploid, and by
subsequent studies of the germinated haploid for marker analysis. The
presence of rnr2-68 allele in haploid was confirmed by
hydroxyurea (HU) sensitivity, benomyl resistance, and arrest in S phase
at 34 °C; whereas cdc33-1 allele was confirmed by
co-segregation of HU resistance at room temperature and G1
arrest at 35 °C. Yeast extract-peptone-adenine-dextrose (YPAD),
yeast extract-peptone-adenine-galactose (YPGal), and synthetic minimal
medium with 2% raffinose, 2% dextrose, or 2% galactose, and
agar-containing plates were prepared as described (28). Plates
containing hydroxyurea or benomyl were made by adding the indicated
amount of hydroxyurea or benomyl to YPAD or YPGal agar after
autoclaving. Yeast transformation was carried out essentially as
described (29) with some modifications.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (65K):
[in a new window]
Fig. 1.
Redistribution of the FGF and ODC transcripts
in the polysomes of CHO-4E. A, Polysomes and Northern
blot of slot-blot fractions. Cell extracts of control
(CHO-BK) and cell extracts overexpressing CHO-4E were
prepared and sedimented for 2 h through sucrose gradients.
Fraction aliquots were applied to a membrane in duplicates with a
slot-blotter. The membrane was cut into strips and probed with either
rat-FGF-2 or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNAs. Fractions corresponding to heavy
polysomes (heavy polys) ( 6 ribosomes); light polysomes
(light polys) (2-5 ribosomes); 80, 60, and 40 S ribosomal
subunits; and small RNPs (Unbound) are indicated. A
graphical representation of the distribution of the particles through
the gradient is also shown. B, Northern analysis of FGF-2,
ODC, and cyclin (Cln)-D1 in P100 and S100. The
centrifugation in this case was extended to 4 h (see under
"Experimental Procedures"). Fractions containing the heavy
polysomes (6) and the rest of the gradient (0-5 ribosomes) were
defined as P100 and S100, respectively. Note the translational
redistribution of FGF-2 and ODC mRNAs in the P100 of CHO-4E cells
(the heavy polysomes). Note also that FGF-2 is expressed as a mixture
of three transcripts, which differ in the length of 3'UTR, by ~1, 2, and 4 kb, respectively.
6 ribosomes. The ribosome pellet, termed
P100, was used to generate the cDNA library. The rest of the
gradient, containing particles with a sedimentation coefficient of five
or fewer ribosomes, was termed S100 and was used as a control in
subsequent experiments. An example of this fractionation is shown in
Fig. 1B, where the distribution of FGF-2 mRNA and ODC,
another eIF4E-dependent mRNA (33), in the P100 and S100 fractions of CHO-BK versus CHO-4E is displayed. The total
amount of FGF-2 or ODC mRNA was the same in CHO-BK and CHO-4E
cells, when S100 and P100 fractions are integrated. However, there is a
reciprocal redistribution of FGF-2 (and ODC) mRNAs in the S100 and
P100 of these cells (Fig. 1B). For comparison, the Northern blot was probed for cyclin-D1 mRNA, which was similar in CHO-BK and
CHO-4E (Fig. 1B, right panel), indicating equivalent
expression and polysomal distribution.
View larger version (48K):
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Fig. 2.
Overexpression of RNR2 in
CHO-4E cells. A, Northern blot of RNR2. The
blot shown in Fig. 1B was probed with one of the isolated
RNR2 clones form the subtracted library. The clone showed
nearly complete identity with RNR2 cDNA of golden
hamster. Note the large redistribution of RNR2 mRNA in
P100. The right panel of A shows that the total
level of the transcript is similar in CHO-BK and CHO-4E. B,
the right panel shows a Western blot of RNR2. The blot
probed with YL1/2 monoclonal antiserum that reacts with -tubulin and
RNR2 (right panel) shows an increase in the level of RNR2 in
CHO-4E. A parallel blot was probed with lactic dehydrogenase
(LDH) (left panel) antibody as a control.
TCDD, tetrachlorodibenzodioxin.
-tubulin.
This antibody recognizes, in addition to tubulin from different
species, a conserved epitope in the C terminus of RNR2 (36). The
Western blot showed a 15-fold increase of the RNR2 protein (42 kDa) in uninduced CHO-4E cells and a 50-fold increase after activation of the
eIF4E promoter with tetrachlorodibenzodioxin (Fig. 2B). This
further confirms that the change in synthesis of RNR2 protein is
largely translational and related to the level of expressed eIF4E. In
contrast to the RNR2 elevation, -tubulin was only modestly increased
(2-fold), possibly an indirect effect of translational autoregulation
(37). Another control protein, lactic dehydrogenase, was equally
expressed in CHO-BK and CHO-4E cells (Fig. 2B, left panel).
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Fig. 3.
CHO-4E and CHO-HU have shorter S phases than
CHO-BK. Control (CHO-BK), CHO-HU, and CHO-4E cells were
synchronized as described under "Experimental Procedures." After
the addition of serum-containing medium (t = 0), DNA
synthesis was measured by pulse-labeling the cells with
[3H]thymidine for 1-h intervals. The mean of three
experiments is plotted; error bars for the S.D. (less than
10% in each case) are not shown in order to avoid crowding.
CHO-HU indicates the cells selected in the medium containing
hydroxyurea.
View larger version (76K):
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Fig. 4.
Overexpression of eIF4E
(CDC33) rescues viability in rnr2-68
strain; cdc33-1 rnr2-68 double mutant shows
conditional synthetic lethality. A, the rnr2-68
ts mutant strain was transformed either with the eIF4E
overexpressing vector (YCpIF15-CDC33, TRP1) or
with the control vector (YCpIF15), and streaked onto a synthetic medium
(without Trp) containing 2% galactose. The plate was incubated at
34 °C for 5 days. B, tetrad dissection of a cross between
congenic rnr2-68 and cdc33-1. The spores from
each ascus were aligned horizontally on a YPAD plate and allowed to
grow at 23 °C for 3-5 days. Note the presence of one and two
small-sized colonies of the double mutant in tetratype and nonparental
ditype tetrads, respectively. C, conditional synthetic
lethality of the double mutant, cdc33-1 rnr2-68, at
30 °C. Colonies from single (parental) and double mutant spores were
streaked onto YPAD plates and incubated at different temperatures for 5 days. One representative plate incubated at 30 °C is shown here.
D, the double mutant cells were incubated at 30 °C, at
which temperature they arrest in S phase as singly budded
(Nomarski, left panel), uninucleated cells (Dapi,
right panel). Dapi, 4',6'-diamidino-2-phenylindole.
Note that the nucleus is alternatively located in the bud or the
mother. DNA content in these arrested cells was shown to be 1 N by propidium iodide staining and flow cytometry (not
shown).
View larger version (46K):
[in a new window]
Fig. 5.
Expression of RNR2 depends
on the level of eIF4E in yeast. A, Northern analysis of
RNR2 mRNA in P100 and S100. Fractions containing the
heavy polysomes ( 6 ribosomes), or the rest of the gradient (0-5
ribosomes) were defined as P100 and S100, respectively. The blot was
probed with a radiolabeled riboprobe of RNR2. Note the
translational redistribution of RNR2 mRNAs in the P100
fractions. The bottom panel was probed with an actin probe
as a loading control. B, Western blot showing the effect of
depletion of functional eIF4E. A temperature shift of the ts
strain cdc33-1, from permissive (25 °C) to restrictive
(37 °C) temperature, shows a drastic reduction in the level of RNR2
protein compared with the control, tubulin. The positions of RNR2 and
-tubulin are indicated. The bottom panel is a Northern
blot of RNA from parallel samples to those analyzed by Western blots.
The bottom panel was probed by RNR1, RNR2, and
actin probes. Note that there were no observable changes in
the levels of these transcripts upon shift to the restrictive
temperature.
-tubulin was unaffected
(Fig. 5B). [35S]Methionine incorporation
showed that the shift to nonpermissive temperature did not result in
gross changes in the total protein synthesis pattern during the first
hour (data not shown). This indicates again that the loss of eIF4E
function results in discreet changes in protein synthesis (like RNR2)
rather than global, at least initially. There were also no observable
changes in the level of RNR2 or RNR1 mRNAs
(Fig. 5B, bottom panel), suggesting that the specific
decrease in the synthesis of RNR2 protein upon reduction of eIF4E
activity occurs at a posttranscriptional level.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Molecular Medicine, Beth Israel
Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave.,
Boston, MA 02215.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Rhoads, R. E.
(1988)
Trends Biochem. Sci.
13,
52-56[CrossRef][Medline]
[Order article via Infotrieve]
2.
Duncan, R.,
Milburn, S. C.,
and Hershey, J. B.
(1987)
J. Biol. Chem.
262,
380-388 3.
De Benedetti, A.,
Joshi-Barve, S.,
Rinker-Schaeffer, C.,
and Rhoads, R. E.
(1991)
Mol. Cell. Biol.
11,
5435-5445 4.
Rhoads, R. E.
(1991)
Curr. Opin. Cell Biol.
3,
1019-1024[CrossRef][Medline]
[Order article via Infotrieve]
5.
Rhoads, R. E.
(1993)
J. Biol. Chem.
266,
3017-3020
6.
Morley, S. J.
(1994)
Mol. Biol. Rep.
19,
221-231[CrossRef][Medline]
[Order article via Infotrieve]
7.
Altmann, M.,
Handschin, C.,
and Trachsel, H.
(1987)
Mol. Cell. Biol.
7,
998-1003 8.
Brenner, C.,
Nakayama, N.,
Goebl, M. G.,
Tanaka, K.,
Toh-e, A.,
and Matsumoto, K.
(1988)
Mol. Cell. Biol.
8,
3556-3559 9.
Lazaris-Karatzas, A.,
Montine, K. S.,
and Sonenberg, N.
(1990)
Nature
345,
544-547[CrossRef][Medline]
[Order article via Infotrieve]
10.
De Benedetti, A.,
and Rhoads, R. E.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
8212-8216 11.
De Benedetti, A.,
Joshi, B.,
Graff, J. R.,
and Zimmer, S. G.
(1994)
Mol. Cell. Differ.
2,
347-371
12.
Kerekatte, V.,
Smiley, K.,
Hu, B.,
Smith, A.,
Gelder, F.,
and De Benedetti, A.
(1995)
Int. J. Cancer
64,
27-31[Medline]
[Order article via Infotrieve]
13.
Nathan, CA,
Liu, L.,
Li, B.,
Nandy, I.,
Abreo, F.,
and De Benedetti, A.
(1997)
Oncogene
15,
579-584[CrossRef][Medline]
[Order article via Infotrieve]
14.
Sonenberg, N.,
and Gingras, A. C.
(1998)
Curr. Opin. Cell Biol.
10,
268-275[CrossRef][Medline]
[Order article via Infotrieve]
15.
De Benedetti, A.,
and Harris, A. L.
(1999)
Int. J. Biochem. Cell Biol.
31,
59-72[CrossRef][Medline]
[Order article via Infotrieve]
16.
Eriksson, S.,
Groppi, V.,
Ullman, B.,
and Martin, D. W., Jr.
(1984)
Adv. Exp. Med. Biol.
165,
407-410
17.
Elledge, S. J.,
and Davis, R. W.
(1987)
Mol. Cell. Biol.
7,
2783-2793 18.
Elledge, S. J.,
and Davis, R. W.
(1990)
Genet. Dev.
4,
740-751
19.
Elledge, S. J.,
Zhou, Z.,
and Allen, J. B.
(1992)
Trends Biochem. Sci.
17,
119-123[CrossRef][Medline]
[Order article via Infotrieve]
20.
Thelander, L.,
and Berg, P.
(1986)
Mol. Cell. Biol.
6,
3433-3442 21.
Zhou, B. S.,
Hsu, N. Y.,
Pan, B. C.,
Doroshow, J. H.,
and Yen, Y.
(1995)
Cancer Res.
55,
1328-1333 22.
Fan, H.,
Villegas, C.,
and Wright, J. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14036-14040 23.
Fan, H.,
Villegas, C.,
Huang, A.,
and Wright, J. A.
(1998)
Cancer Res.
58,
1650-1653 24.
Jensen, R. A.,
Page, D. L.,
and Holt, J. T.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9257-9261 25.
Bonneau, A. M.,
and Sonenberg, N.
(1987)
J. Biol. Chem.
262,
11134-11139 26.
Kevil, C.,
Carter, P.,
Hu, B.,
and De Benedetti, A.
(1995)
Oncogene
11,
2339-2348[Medline]
[Order article via Infotrieve]
27.
Raue, H. A.,
Mager, W. H.,
and Planta, R. J.
(1991)
Methods Enzymol.
194,
453-455[Medline]
[Order article via Infotrieve]
28.
Sherman, F.
(1991)
Methods Enzymol.
194,
3-21[CrossRef][Medline]
[Order article via Infotrieve]
29.
Gietz, D.,
Jean, A.,
St,
Woods, R. A.,
and Schiestl, R. H.
(1992)
Nucleic Acids Res.
20,
1425 30.
Adams, A.,
Gottschling, D. E.,
Kaiser, C. A.,
and Stearns, T.
(1997)
Methods in Yeast Genetics
, pp. 193-194, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
31.
Allen, J. B.,
Zhou, Z.,
Siede, W.,
Friedberg, E. C.,
and Elledge, S. J.
(1994)
Genes Dev.
8,
2416-2428 32.
Lodish, H. F.
(1974)
Nature
251,
385-388[CrossRef][Medline]
[Order article via Infotrieve]
33.
Graff, J. R.,
De Benedetti, A.,
Olson, J. W.,
Tamez, P.,
Casero, R. A., Jr.,
and Zimmer, S. G.
(1997)
Biochem. Biophys. Res. Commun.
240,
15-20[CrossRef][Medline]
[Order article via Infotrieve]
34.
Kevil, C.,
De Benedetti, A.,
Payne, K. D.,
Coe, L. L.,
Laroux, S.,
and Alexander, S.
(1996)
Int. J. Cancer
65,
785-790[CrossRef][Medline]
[Order article via Infotrieve]
35.
Chaudhuri, M. M.,
Tonin, P. N.,
and Srinivasan, P. R.
(1992)
Biochim. Biophys. Acta
1171,
117-121[Medline]
[Order article via Infotrieve]
36.
Standart, N.,
Hunt, T.,
and Ruderman, JV.
(1986)
J. Cell Biol.
103,
2129-2136 37.
Yen, T. J.,
Gay, D. A.,
Pachter, J. S.,
and Cleveland, D. W.
(1988)
Mol. Cell. Biol.
8,
1224-1235 38.
DeFatta, R. J.,
Turbat-Herrera, E. A.,
Li, B. D. L.,
Anderson, W.,
and De Benedetti, A.
(1999)
Int. J. Cancer
80,
518-522
39.
Pelletier, J.,
and Sonenberg, N.
(1987)
Biochem. Cell Biol.
65,
576-581[Medline]
[Order article via Infotrieve]
40.
Kozak, M.
(1989a)
Nucleic Acids Res.
15,
8125-8147 41.
Kozak, M.
(1989b)
J. Cell Biol.
108,
229-241 42.
Geballe, A. P.,
and Morris, D. R.
(1994)
Trends Biochem. Sci.
19,
150-164
43.
Ruan, H.,
Shantz, L. M.,
Pegg, A. E.,
and Morris, D. R.
(1996)
J. Biol. Chem.
271,
29576-29582 44.
Amara, F. M.,
Chen, F. Y.,
and Wright, J. A.
(1995)
Nucleic Acids Res.
23,
1461-1467 45.
Wells, S. E.,
Hillner, P. E.,
Vale, R. D.,
and Sachs, A. B.
(1998)
Mol. Cell
2,
135-140[CrossRef][Medline]
[Order article via Infotrieve]
46.
Fernandez-Sarabia, M. J.,
McInerny, C.,
Harris, P.,
Gordon, C.,
and Fantes, P.
(1993)
Mol. Gen. Genet.
238,
241-251[Medline]
[Order article via Infotrieve]
47.
Huang, M.,
and Elledge, S. J.
(1997)
Mol. Cell. Biol.
17,
6105-6113[Abstract]
48.
Wang, P. J.,
Chabes, A.,
Casagrande, R.,
Tian, X. C.,
Thelander, L.,
and Huffaker, T. C.
(1997)
Mol. Cell. Biol.
17,
6114-6121[Abstract]
49.
Moore, S. A.
(1988)
J. Biol. Chem.
263,
9674-9681 50.
Zhao, X.,
Muller, E. G.,
and Rothstein, R.
(1998)
Mol. Cell.
2,
329-340[CrossRef][Medline]
[Order article via Infotrieve]
51.
Ali, M. A.,
McWeeney, D.,
Milosavljevic, A.,
Jurka, J.,
and Jariwalla, R. J.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
8257-8261 52.
Huang, A.,
Fan, H.,
Taylor, W. R.,
and Wright, J. A.
(1997)
Cancer Res.
57,
4876-4881 53.
Kulsh, J.
(1997)
Med. Hypotheses
49,
297-300[CrossRef][Medline]
[Order article via Infotrieve]
54.
Rozell, B.,
Stenman, G.,
Magnusson, B.,
Lekholm, U.,
Nagle, R. B.,
and Hansson, H. A.
(1986)
J. Oral Pathol.
15,
261-264[CrossRef][Medline]
[Order article via Infotrieve]
55.
Sorells, D. L.,
Gahli, E. G.,
Meschonat, C.,
DeFatta, R. J.,
Black, D.,
Li, L.,
De Benedetti, A.,
Nathan, C. A.,
and Li, B. D. L.
(1998)
Head Neck
21,
60-65[CrossRef]
56.
Kohrer, K.,
and Domdey, H.
(1991)
Methods Enzymol.
194,
398-405[Medline]
[Order article via Infotrieve]
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