From the Division of Basic Sciences and
¶ Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI,
National Institutes of Health, Bethesda MD 20892,
Greenbaum
Cancer Center, University of Maryland, Baltimore MD 21201, ** Department
of Molecular Genetics and Microbiology, University of Medicine and
Dentistry of New Jersey (UMDNJ), Robert Wood Johnson Medical School and
The Graduate Programs in Molecular Bioscience Rutgers/UMDNJ,
Piscataway, New Jersey 08854, and
Laboratory of Biological Chemistry, NIA,
National Institutes of Health, Baltimore, Maryland 21224
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ABSTRACT |
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We report the cloning and characterization of a
DNA damage-inducible (DDI) transcript DDI A121. The full-length human
DDI A121 cDNA contains an open reading frame of 113 amino acids,
corresponding to a protein of 12.7 kDa. The deduced amino acid sequence
of A121 shows high homology to the yeast translation initiation factor (eIF) sui1 and also exhibits perfect identity to the partial sequence of recently purified human eIF1. Expression of human A121 corrected the
mutant sui1 phenotype in yeast, demonstrating that human A121 encodes a
bona fide translation initiation factor that is equivalent to yeast sui1p. The mammalian A121/SUI1 gene exhibits two
transcripts (1.35 kilobases and 0.65 kilobases) containing a common
coding region but differing in their 3'-untranslated region. The long and short A121/SUI1 mRNAs are differentially regulated by genotoxic and endoplasmic reticulum stress. The genotoxic stress induction of
A121/SUI1 mRNA is conserved in both humans and rodents and occurs
in a p53-independent manner. Our identification of a stress-inducible cDNA that encodes eIF1 suggests that modulation of translation initiation appears to occur during cellular stress and may represent an
important adaptive response to genotoxic as well as endoplasmic reticulum stress.
DNA damage (genotoxic stress) can be induced by exogenous agents
including radiation and chemicals or by endogenous processes involving
the generation of reactive oxygen species (reviewed in Ref. 1).
Cellular response to genotoxic stress is complex and affects a variety
of cellular processes including cell cycle progression, replication,
transcription, signal transduction, DNA repair, mutagenesis, and
apoptosis (reviewed in Refs. 1 and 2). A number of different genes have
been identified which are believed to affect these cellular processes
in response to genotoxic stress (reviewed in Ref. 1). Low ratio
hybridization subtraction has been previously used in this laboratory
to isolate and clone a number of genotoxic stress-inducible genes
including five GADD (growth arrest and
DNA damage-inducible) genes (3, 4). GADD45 is a
p53-regulated gene that appears to play a role in genomic
stability1; GADD153 encodes a
small protein of the C/EBP family of transcription factors (5); GADD34
codes for a protein that shows homology to a herpes simplex viral
protein and appears to play a role in apoptosis (6). The hamster
GADD33 gene is equivalent to the human cornifin gene that is
involved in keratinocyte differentiation (1), whereas GADD7 does not
appear to code for a protein product (7). The expression of the
GADD genes has been shown to be regulated by a number of
genotoxic and nongenotoxic stresses (Ref. 8 and references therein).
In addition to GADD genes, the cDNAs representing
p21WAF1, proliferating cell nuclear antigen, initiation factor
5 (eIF5),2 and thrombomodulin
genes were also detected as genotoxic stress-inducible transcripts in
the same enriched library (8). In an attempt to identify and clone the
genes corresponding to the remaining novel genotoxic stress-inducible
transcripts present within the same library, we have recently cloned
and characterized a novel genotoxic stress-inducible gene (8). This
gene, named A18 heteronuclear ribonucleoprotein
(18hnRNP) is specifically regulated by UV and UV-mimetic agents and encodes a putative glycine-rich RNA and DNA-binding protein (8). In the current study, we report the cloning
and characterization of another human gene that corresponds to one of
the previously uncharacterized genotoxic stress-inducible transcripts
named A121. The full-length cDNA of human A121 encodes translation
eIF1, showing a high homology to the yeast translation initiation
factor sui1.
Cell Lines--
The following cell lines were used in this
study: MCF-7, breast carcinoma cells; RKO, colon carcinoma cells; A549
and H1299 lung carcinoma cells; Sk-N-SH, neuroblastoma cells; OVCAR,
ovarian carcinoma cells; HL60, promyelocytic leukemia cells; ML-1,
myeloid leukemia cells; WMN, Burkitt's lymphoma cells; GM536,
lymphoblastoma cells.
Cell Treatment--
Cellular exposure to UV or ionizing
radiation and treatment with MMS were as described previously (4, 9).
For treatment with thapsigargin (LC Laboratories) and
2,5-di-tert-butylhydroquninone (DBHQ) (Sigma), exponentially
growing cells were treated with 2 µM thapsigargin or
50 µM DBHQ for various time points, and cells were
harvested for RNA extraction.
DDI Library Construction and Sequencing--
The low ratio
hybridization subtraction procedure and construction of the original
DDI library have been described previously (4). In brief, Chinese
hamster ovary cells in logarithmic phase of growth were exposed to 14 J/m2 UV, and 4 h later, poly (A)+ RNA was
extracted. Poly(A)+ RNA from UV-irradiated cells was
reverse-transcribed into single-stranded cDNAs, which were then
hybridized at a high Rot with poly(A)+ RNA from
unirradiated cells. The single-stranded cDNA was obtained from
cDNA:RNA hybrids after alkali treatment and hybridized to the
original irradiated poly(A)+ RNA, and the resulting DNA:RNA
duplexes were obtained using a hydroxylapatite column. Single-stranded
DNA was isolated from the cDNA:RNA duplex and subsequently used for
second strand synthesis. The double-stranded cDNAs were subcloned
into a plasmid pXF3 using a GC-tailing method (3, 4). The
partial-length clones in the DDI library were sequenced by the dideoxy
chain termination method as described previously (4).
cDNA Cloning of Human A121--
The hamster DDI A121 clone
was 298 base pairs long; a human EST clone 117124 (accession number
T87717) showing high homology to the nucleotide sequence of hamster DDI
A121 was obtained from IMAGE Consortium, LLNL (Lawrence Livermore
National Laboratory) and sequenced in its entirety. The sequence
information from the EST clone revealed the presence of a
polyadenylation signal and a poly(A) tail, suggesting that the EST
clone 117124 corresponded to the 3'-end of the gene. Based on the EST
sequence information, we synthesized primers corresponding to the most
5'-end of the EST sequence to amplify the remaining 5'-end of the
cDNA from a human placenta Marathon-Ready cDNA library
(CLONTECH, Palo Alto, CA). This library contains
adaptor-ligated double-stranded cDNA and the adaptor sequence
serves as the 5' or 3' primer. The adaptor sequence specific primer
5'-CCATCCTAATACGACTCACTATAGGGC-3' was used as 5'-primer, and the
gene-specific primer 5'-CTATCACCTCGCTAGCGG-3' was used as 3'-primer in
the amplification reactions. The amplification conditions were as
follows: one cycle of 1 min at 94 °C; 25 cycles of 1 min at
94 °C, 1 min at 60 °C, and 4 min at 68 °C; and then 1 cycle of
4 min at 68 °C. The amplified products were gel-purified and cloned
into a TA-cloning vector (CLONTECH, Palo Alto, CA). Several independent clones were analyzed for the presence of the amplified inserts, and the two largest cDNA clones representing the
5'-end of human A121 were sequenced in their entirety. Both of these
clones, which were otherwise identical, differed in length from each
other at their 5'-ends by 107 nucleotides and were collinear at their
3'-ends with the EST clone 117124. Homology searches using
GenBankTM/EBI and EST data banks identified a number of EST clones
exhibiting nucleotide sequence corresponding to the coding region of
our human A121 cDNA. These EST sequences have also been aligned to
obtain a computer-generated open reading frame (10).
Northern and Dot Blot Hybridizations and cDNA
Probes--
RNA extraction and Northern and quantitative dot blot
hybridizations were performed as we have described previously (11). A
phosphoimager, Yeast Strains, Media, Plasmid Constructions, and Genetic
Procedures--
The following strains of Saccharomyces
cerevisiae were used in this study: Y157, MAT Molecular Cloning of Human A121--
As recently described (8),
the cDNAs representing the DDI transcripts in the original hamster
library were only partial length and ranged in size from 0.2-0.5
kilobases. These cDNAs were sequenced, and the sequence information
was used to search the sequence data banks. The partial-length sequence
of the original hamster DDI clone named A121 exhibited high homology
(84.3%) to several human EST sequences. These EST clones did not show
homology to the sequences of known genes deposited in the public data
banks, suggesting that they represented a potentially novel gene. The sequence information from EST clone 117124 (accession number T87717) was used to screen a human placenta cDNA library as described under
"Experimental Procedures." The complete nucleotide sequence of the
full-length cDNA of human DDI A121 is shown in Fig.
1. The 1312-nucleotide-long human A121
cDNA contains an open reading frame predicted to code for a
113-amino acid-long protein of 12.7 kDa. The BLASTX alignment of the
predicted protein sequence of the human A121 cDNA with other
protein sequences in the data banks revealed that human A121 is
homologous to a less well characterized yeast translation initiation
factor named sui1 (16). In Fig. 2, the
deduced amino acid sequence of human A121 is aligned with the predicted
amino acid sequences from various species. As is shown, human A121
(hereafter referred to as A121/SUI1) shows a high degree of homology to
the corresponding proteins from other species including mouse, chick,
mosquito, flower, and yeast.
Molecular Characterization of Human A121/SUI1
Transcripts--
A121/SUI1 gene displayed two major
transcripts of ~1.35 and ~0.65 kilobases in size, with some
heterogeneity occurring in their expression in various tissues (Fig.
3). The two A121/SUI1 transcripts could
result from alternative splicing or from the differential usage of
polyadenylation signals. Sequence analysis of the 3'-untranslated
region of the human A121/SUI1 cDNA revealed the presence of two
polyadenylation signals, suggesting that the latter possibility was
more likely. To further investigate this issue, we performed Northern
analyses using two cDNA probes corresponding to the different
regions of human A121/SUI1 cDNA. Probe A (Fig. 3) corresponds to
the entire open reading frame and part of the 3'-untranslated sequence
up to the first polyadenylation signal, whereas probe B (Fig. 3)
represents the 3'-untranslated sequence downstream of the first
polyadenylation signal. The results of Northern analyses shown in Fig.
3 demonstrate that probe A detects two transcripts, whereas probe B
detects only the larger 1.35-kilobase transcript on the same blots;
this was true in both human and mouse tissues. These results highlight
that indeed the two A121/SUI1 transcripts differ from each other at
their 3'-untranslated region and most likely result because of the
alternative use of polyadenylation signals. These results also explain
why the hamster cDNA probe, which correspond to the 3'-untranslated
region, detected only the larger transcript. Because the human EST
sequences correspond to the 3'-untranslated region of human A121/SUI1
and not to the coding region, the above findings may also explain why
the former did not display homology to sui1 from yeast and other
species.
Genotoxic and Endoplasmic Reticulum (ER) Stress Regulation of Human
A121/SUI1--
The expression of hamster A121 transcripts was induced
by the genotoxic agents UV, UV mimetic agents, and MMS in Chinese
hamster ovary cells; a number of other agents, by contrast, did not
modulate A121 expression in hamster cells. Table
I summarizes the overall effects of
various stress-inducing agents on A121 mRNA regulation in Chinese
hamster ovary cells. We next sought to investigate the regulation of
human A121/SUI1 in response to genotoxic and nongenotoxic stress in
human cells. Our results demonstrated that human A121/SUI1 mRNA was
induced by MMS in all the cell lines tested, whereas its induction in
response to UV was cell type-specific. Ionizing radiation (
MMS up-regulates only the larger A121 transcript and not the smaller
transcript (see below), suggesting that MMS appears to mediate A121
regulation via elements that reside in the 3'-untranslated region. We
next investigated the effects of the transcription inhibitor
actinomycin D and the protein synthesis inhibitor cycloheximide on the
MMS induction of human A121/SUI1 mRNA. As shown in Fig. 4, actinomycin D blocked the MMS
induction of A121/SUI1 mRNA expression in MCF-7 cells;
cycloheximide alone, by contrast, enhanced the mRNA levels of
A121/SUI1 and further potentiated the MMS effect on A121/SUI1 mRNA
levels. These results suggest that new protein synthesis is not
required for the MMS-mediated increase in the A121/SUI1 mRNA
levels. These results also suggest that the constitutive A121/SUI1
mRNA levels appear to be negatively regulated by certain labile
protein factors. Similar experiments were also performed in Chinese
hamster ovary cells, and identical results were obtained (data not
shown).
We next sought to determine whether the expression of A121/SUI1 can
also be modulated by conditions that primarily cause ER stress.
Thapsigargin and DBHQ are specific inhibitors of
sarcoplasmic/endoplasmic reticulum Ca2+ transport
ATPases. By inhibiting sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPases, these agents block the re-uptake of
cytosolic Ca2+ into the ER, resulting in depletion of
Ca2+ from the ER Ca2+ storage compartments.
Stress inflicted upon cells by depletion of these Ca2+
pools can have profound effects on cell growth (18) and can lead to
certain adaptive responses (17-19). Treatment of MCF-7 cells with
either thapsigargin or DBHQ for 4 and 24 h results in induction of
A121/SUI1 mRNA levels when compared with untreated cells (Fig.
5), and the degree of increase in the
A121/SUI1 mRNA levels appear to parallel that seen with MMS (Fig.
5). These data suggest that the induction of A121/SUI1 may represent a
stress response that occurs when cells are exposed to injury (genotoxic or otherwise). It is also of note that unlike MMS and UV irradiation (see below), which up-regulate only the larger transcript, the ER
stress-inducing agents up-regulate both transcripts (Fig. 5), suggesting that the potential mechanisms of A121/SUI1 mRNA
regulation by genotoxic stress and ER stress might be different.
Genotoxic Stress Induction of A121/SUI1 Is Conserved in Mouse and
Occurs in a p53-independent Manner--
The levels of wild-type p53
protein are enhanced by genotoxic agents such as ionizing radiation,
UV, and MMS (Ref. 11 and references therein). p53 in turn
transcriptionally up-regulates the expression of its downstream
effector genes (11 and references therein). To ascertain whether
A121/SUI1 is a p53-regulated gene, we investigated the
genotoxic stress regulation of A121 in p53 wild-type and p53 knock-out
cells. Primary keratinocytes obtained from p53 wild-type and
p53-knock-out mice were exposed to MMS and UV irradiation, and the
effect on A121/SUI1 expression was investigated. As shown in Fig.
6, MMS and UV induction of A121/SUI1 mRNA was noted in both genotypes, and the lack of p53 did not considerably affect the extent of A121/SUI1 mRNA induction. Again, the data in Fig. 6 show that both MMS and UV irradiation predominantly up-regulate the larger A121/SUI1 transcript. These results demonstrate that the genotoxic stress regulation of A121/SUI1 mRNA is conserved in humans and rodents and that it occurs in a p53-independent manner.
Human A121 Appears to Encode Human eIF1 and Can Correct the Mutant
sui1 Phenotype in Yeast--
Pestova et al. (20) recently
purified and partially sequenced the human eIF1 and eIF1A proteins. The
sequence (GDDLLPAGT and TLTTVQGIA) of two tryptic peptides representing
human eIF1 show exact identity to the corresponding amino acid sequence
predicted for A121 protein. The molecular mass (13 kDa) of purified
eIF1 is also similar to the predicted molecular mass (12.7 kDa) of the
A121 protein product. Human eIF1 and eIF1A were reported to have
distinct yet synergistic activities that were required for recognition
of the initiation codon (20). Human eIF1 alone has been reported to be
necessary for ensuring the fidelity of translation initiation by
recognizing and destabilizing the incorrectly assembled ribosomal
complexes at the initiation codon. Similar function has been reported
for yeast sui1p. Deletion of the sui1 gene is lethal (16,
21); temperature-sensitive mutations within the sui1 gene
have been identified that do not allow the mutant strains to grow at
37 °C (13, 16, 21). The temperature-sensitive sui1 mutations also
allow translation to initiate at UUG start codon (16, 21, 13).
Introduction of the exogenous wild-type sui1 gene has been
shown to inhibit translation of
his4UUG-lacZ in sui1 mutant
background (13, 16, 21). Furthermore, sui1 mutant strains carrying the
exogenous wild-type sui1 gene were able to grow at both
permissive and nonpermissive temperatures (13, 16, 21).
We reasoned that if A121/SUI1 cDNA encodes a bona fide
translation initiation factor equivalent to yeast sui1p, then human A121/SUI1 should function in yeast and correct mutant sui1 phenotype. To test this contention, we subcloned the human A121 cDNA into a
yeast single copy expression vector pG-1 (13, 14) and tested its
ability to suppress translation of
his4UUG-lacZ gene. The reporter
vector his4UUG-lacZ contains the
His4 gene fused in-frame with the lacZ gene, and
the AUG start codon of the His4 gene is replaced with UUG as
a start codon (16, 21, 13). Various yeast strains carrying the plasmid
borne human A121(pJD176F) or expression plasmid without A121 insert
(pG-1) were transformed with the reporter vector
his4UUG-lacZ, and the
Previously, we have reported the cloning and characterization of
several important genotoxic stress-inducible genes from the hamster DDI
library originally made using a low ratio hybridization subtraction
technique (Refs. 3, 4, and 8 and references therein). The clones in
this library represent transcripts that are induced within 4 h
after exposure to UV irradiation (4). Here we report the identification
and molecular cloning of the full-length cDNA of a previously
uncharacterized DDI A121 transcript from the same library. The deduced
amino acid sequence of human A121 shows high homology to the yeast
translation initiation factor sui1 (16) and the corresponding proteins
from other species including mouse (direct submission, accession number
P48024), chick (23), mosquito (24), and Arabadopsis thaliana
(direct submission, accession number P41568). The tissue distribution of A121/SUI1 mRNA in human and mouse tissues revealed that
A121/SUI1 is expressed in most tissues; whether A121/SUI1 is absolutely required for protein translation in mammalian cells or is dispensable must await the results of A121/SUI1 gene knock-out studies.
Yeast sui1 has been shown to be an essential gene (16, 21) and was originally identified as a translation initiation suppressor locus (21). In addition to sui1, two other genes, namely sui2 and SUI3, have also been identified in yeast (21, 22). Mutations at all these three unlinked loci restored expression of the HIS4 allele
lacking the canonical AUG translation initiation start site (21), a
finding that implicates their role in controlling the fidelity of
translation initiation (21).
Unlike sui1, the sui2 and SUI3 genes
have been reported to encode the The A121/SUI1 gene exhibits two transcripts of varying
abundance in different tissues. Using cDNA probes corresponding to different regions of A121/SUI1, we have demonstrated that these transcripts result from alternative usage of two poly(A) signals present in the 3'-untranslated region. It is interesting that both
transcripts are differentially regulated by genotoxic and ER stress.
For example, the genotoxic agents MMS and UV induced expression of only
the larger transcript, whereas ER stress-inducing agents up-regulate
both transcripts. These results would suggest that genotoxic stress
either exerts a transcriptional influence or enhances the A121/SUI1
mRNA stability via elements residing within the 3'-untranslated
region. ER stress-inducing agents, on the other hand, may
transcriptionally up-regulate the levels of A121/SUI1 transcripts from
the common 5'-end. Future studies will illuminate the molecular basis
for the differential regulation of A121/SUI1 transcripts by genotoxic
and ER stress-inducing agents.
Although the transcripts encoding human eIF5 were also present in the
original DDI library, eIF5 mRNA expression was not induced by MMS
and only minimally induced by UV in some human cell lines (8). eIF1A is
the interacting partner of human eIF1; however, eIF1A expression was
not regulated by genotoxic or ER stress (data not shown). From the
results presented in this study, it is evident that (a) only
the expression of A121/SUI1 is regulated by genotoxic and ER stress,
whereas that of eIF1A is not, (b) the A121/SUI gene expression is regulated in an agent-specific manner, and (c) its induction is not a general cellular response to all
types of stresses. Because of the lack of the availability of
antibodies against eIF1, we were unable to test its regulation at the
protein levels. Conceivably, genotoxic and ER stress induction of
A121/SUI1 mRNA and protein is coupled and may represent part of the
defense mechanism of a cell to provide a translational checkpoint
control to modulate the expression of certain proteins at the
translational level. Further studies are needed to test this contention
and should lead to a better understanding of the process of translation initiation during various cellular stresses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-scope, and a light densitometer were used to quantitate the signals. The cDNA probes used in this study include a hamster 298-base pair A121 cDNA clone corresponding to the
3'-untranslated region and a human A121 cDNA clones corresponding
to the complete open reading frame and the 3'-untranslated region. For
human eIF1A cDNA probe, a human EST clone (380394) carrying the
human eIF1A cDNA was obtained from IMAGE consortium, LLNL.
leu2 trp1
his4-303(AUU) ura3-52::his4(AUU)-lacZ sui1-1; Y158,
MAT
leu2 trp1 his4-303(AUU)
ura3-52::his4(AUU)-lacZ sui2-1; Y244, MAT
leu2 trp1 his4-303(AUU) ura3-52::his4(AUU)-lacZ SUI3-3; Y159, MAT
leu2 trp1 his4-303(AUU)
ura3-52::his4(AUU)-lacZ. YPAD and synthetic complete
medium lacking tryptophans (H-trp) were as previously reported (12,
13). Temperature sensitivity of yeast strains was confirmed by plating
them onto (H-leu, -trp) medium and by testing their growth at 30 or
37 °C. The yeast expression vector pG-1 (14) carries the yeast
glyceraldehyde-3-phosphate dehydrogenase promoter and the
phosphatidylglycerol kinase 1 terminator sequences. Human A121 cDNA
carrying the entire open reading frame and the 3'-untranslated region
up to the first poly(A) signal was subcloned into pG-1 under the
control of the glyceraldehyde-3-phosphate dehydrogenase promoter to
create pJD176F. Transformation of yeast and Escherichia coli
were performed as described previously (12, 13).
-Galactosidase
activity assays were performed in triplicate as previously reported
(12, 13, 15).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The cDNA and deduced amino acid sequences
of the human A121 gene. The two polyadenylation
signals are underlined.
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Fig. 2.
Amino acid sequence alignment of human
A121/SUI1 with the corresponding amino acid sequences from other
species. GenBankTM accession number and reference for each
sequence are as follows: chick, P51971 (23); mouse, P48024 (direct
submission); Anopheles gambiae, P42678 (24);
Arabadopsis P41568 (direct submission); yeast, P32911 (16).
Chick and mouse sequences are partial-length, and the dashes
represent gaps that were introduced to achieve better sequence
alignment.
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Fig. 3.
Molecular characterization of the two
A121/SUI1 transcripts of A121/SUI1 in various human tissues. A
Northern blot (CLONTECH) containing
poly(A)+ RNA from various human organs was hybridized with
human A121/SUI1 probe (Probe A), corresponding to the coding
region and part of the 3'-untranslated region up to the first
polyadenylation signal (upper panels). The same blot was
also probed with the human A121/SUI1 probe (Probe B),
corresponding to the 3'-untranslated region downstream of the first
polyadenylation signal (lower panels). S. Muscle,
skeletal muscle; kb, kilobases; ORF, open reading
frame; UTR, untranslated region.
-rays, 5 to 20 gray), on the other hand, did not regulate human A121/SUI1
expression in any of the human cell lines tested. The overall effects
of MMS and UV on A121/SUI1 mRNA expression in the various cell
lines are outlined in Table II.
A121 mRNA regulation by different stress-inducing agents in
Chinese hamster ovary cells
MMS and UV regulation of human A121/SUI1 mRNA in various human
cancer cell lines
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Fig. 4.
Effects of transcription inhibitor
actinomycin D (Act D) and protein synthesis inhibitor
cycloheximide (CHX) on MMS induction of human
A121/SUI1 mRNA. MCF-7 human breast carcinoma cells in the
logarithmic phase of growth were treated with MMS (100 µg/ml),
actinomycin D (4 µg/ml), or cycloheximide (50 µg/ml) for various
periods of time as shown. Some of the plates were also treated with a
combination of MMS and actinomycin D or MMS and CHX (actinomycin D or
CHX was added 1 h before the addition of MMS). RNA extraction and
Northern blot hybridization were performed as described under
"Experimental Procedures." Blots were probed with a human A121/SUI1
cDNA fragment representing the 3'-untranslated region. The
experiment was repeated, and similar results were obtained.
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Fig. 5.
A, a Northern blot showing the effects
of endoplasmic reticulum stress-inducing agents on human A121/SUI1
mRNA levels. Cells were either left untreated or treated with the
indicated agents for 4 and 24 h. The effect of MMS on A121/SUI1
mRNA induction is also shown for comparison. A representative blot
was probed with a A121/SUI1 cDNA corresponding to the entire open
reading frame. The schematic representation of the 5'-end and 3'-end
probes is shown in Fig. 3. The experiments were performed twice, and
similar results were obtained. TG, thapsigargin.
B, quantitation of MMS, thapsigargin, and DBHQ-induced
A121/SUI1 mRNA levels. Signals were quantitated as described under
"Experimental Procedures," and the values are plotted relative to
untreated control, which was given a value of 1. The upper
panel shows the relative expression of the larger transcript,
whereas the lower panel shows the relative expression of the
smaller transcript.
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Fig. 6.
MMS and UV irradiation increase murine
A121/SUI1 mRNA levels in a p53-independent manner. Primary
keratinocytes were obtained from p53 wild-type (WT) and p53
null mice as described previously (26). Cells were either left
untreated or treated with MMS (50 and 100 µg/ml) for approximately
18 h (A) or exposed to 50 J/m2 UV
irradiation and harvested 6 h later (B). Total RNA was
extracted, and Northern blot hybridization was performed as described
under "Experimental Procedures." A human A121/SUI1 cDNA probe
corresponding to the entire open reading frame was used to probe the
blots. Quantitation of the representative blots shown was performed as
described under "Experimental Procedures," and the values were
plotted relative to untreated controls.
-galactosidase activities were determined. From the results
summarized in Table III, it is clear that
human A121/SUI1 suppresses
his4UUG-lacZ
-galactosidase
activity only in the sui1 strain and not in the sui2 and SUI3 mutant
strains. sui2 and SUI3 encode the
and
subunits of eIF2,
respectively (22), and mutations in both sui2 and SUI3 also allow
translation to initiate at the UUG codon, although only sui1 and sui2
mutations confer growth related temperature sensitivity (16, 21, 22).
The plasmid expressing human A121/SUI1 only allowed the sui1-1 strain
and not the sui2-1 to grow at both permissive and nonpermissive
temperatures (Table IV). The ability of
human A121/SUI1 to correct only the sui1 phenotype and not the sui2 and
SUI3 phenotypes not only highlights the specificity of A121/SUI1
function but also demonstrates that human A121/SUI1 can function in
yeast.
When expressed in yeast the A121 cDNA clone (pJD176F) complements
the sui1-1 allele but not sui2-1 or SUI3-3
When expressed in yeast, the A121 cDNA clone (pJD176F) allows
growth at both permissive and nonpermissive temperature only in sui1-1
background and not in sui2-1 background
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of eIF2,
respectively (22). Although sui1p has been reported to enhance the
function of eIF3, sui1p may not be a bona fide subunit of
eIF3 (25). Recently Cui et al. (13) reported the
characterization of a novel allele of sui1 named mof2-1. The
sequence of the wild-type mof2 gene was identical to
that of sui1, and both were able to independently correct
mof2-1 or sui1 phenotypes and ensure translation fidelity (13).
The fact that the partial amino acid sequence of the recently purified
human eIF1 (20) is identical to the predicted amino acid sequence of
A121/SUI1 suggests that the latter is likely to encode human eIF1. It
has recently been reported that human eIF1 and eIF1A act in concert to
promote 48 S ribosomal complex formation at the initiation codon (20);
43 S complex lacking eIF1 and eIF1A does not reach the initiation codon
(20). Furthermore, eIF1 alone was able to recognize and destabilize the
aberrantly formed complexes at the initiation codon, a function that is
consistent with that reported for yeast sui1 (16, 13). Our findings
that expression of human A121/SUI1 in yeast was able to correct the sui1 but not the sui2 or SUI3 phenotypes demonstrate that A121/SUI1 encodes the human equivalent of yeast sui1p.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF100737 and F100738.
§ To whom correspondence should be addressed: DBS, NCI, National Institutes of Health, Bldg. 37, Rm. 5C09, Bethesda MD 20892. Tel.: 301-402-0745; Fax: 301-480-2514; E-mail: mssheikh{at}box-m.nih.gov.
1 A. J. Fornace, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic translation initiation factor; MMS, methylmethane sulfonate; DDI, DNA damage-inducible; EST, expressed sequence-tagged; DBHQ, 2, 5-di-tert-butylhydroquninone; ER, endoplasmic reticulum.
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