From INSERM U463, Institut de Biologie-CHR, 9 Quai Moncousu, 44093 Nantes Cedex 1, France
Received for publication, December 5, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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TIA-1 and TIAR are a pair of related RNA-binding
proteins which have been implicated in apoptosis. We show that chicken
DT40 cells with both tia-1 alleles and one tiar
allele disrupted (tia-1( TIA-1 (1 ,2) and the TIA-1-related protein TIAR (3) are a pair of
similar, ubiquitous RNA-binding proteins with three RNA recognition
motifs (RRMs).1 These
proteins fill important functions in both the cytoplasm and the
nucleus, as do a number of other multifunctional regulatory proteins
(4). In the cytoplasm, TIA-1 and TIAR control translation of some
specific mRNAs (5-9) and are active in the general translational arrest mechanism induced by stress (10-15). TIA-1 and TIAR shuttle between cytoplasm and nucleus, but are predominantly nuclear in most
cells studied. In the nucleus, they activate splicing of exons with
weak 5'-splice sites followed by uridylate-rich stretches (16-18).
TIA-1 and TIAR appear to bind to these stretches (they both bind
preferentially to uridylate-rich sequences in vitro, Ref.
19) and assist binding of U1 snRNP to the adjacent 5'-splice site.
Possible target exons include an fgfr-2 alternative exon (16), a Drosophila msl-2 exon (20), a human fas
exon (20), and some alternative human tia-1 and
tiar exons containing premature stop codons (21). TIAR and
possibly TIA-1 are also involved in replication of the RNA genomes of
West Nile virus (22).
Overexpression of TIA-1 or TIAR induces apoptosis, whether accomplished
by exposure of permeabilized cells to recombinant proteins (1, 3), or
by infecting cells with an engineered virus (23). During Fas-induced
apoptosis, TIA-1 is phosphorylated (24), and TIAR is relocated to the
cytoplasm (25). TIA-1 activates splicing of a fas
pre-mRNA exon so as to favor production of a cell death receptor,
at the expense of a soluble form of Fas (17). Based on these
observations, TIA-1 and TIAR have been proposed to be
apoptosis-promoting proteins (17, 20). On the other hand, ~50% of
tia-1( One way to do so was to disrupt the genes in a cell line. Homologous
recombination is very efficient in the chicken B-cell line DT40 (27,
28), and this line has been used extensively for gene disruption
studies (29-31). Using a gene disruption approach, we show here that
DT40 cells expressing only TIA-1 or only TIAR are viable. However, we
were unable to obtain cells expressing neither of the two proteins,
suggesting a requirement for at least one of the TIA-1/TIAR pair for
cell viability. We also show that elements of a tiar gene
auto-control mechanism are highly conserved between chickens and humans
and discuss whether TIA-1/TIAR should be regarded as
apoptosis-promoting factors or cell survival factors.
Cloning of Chicken tia-1 and tiar Genes--
Chicken TIA-1 and
TIAR ESTs were identified by searching a data base (32) with the human
TIA-1 and TIAR cDNA sequences. The corresponding TIA-1 cDNA
clone (DKFZp426G159) was obtained from the Resource Center of the
German Human Genome Project, Max-Planck-Institute for Molecular
Genetics, Berlin, and sequenced. It coded for a truncated TIA-1,
containing a deletion of 5 base pairs (AGCAG) at an
exon-exon junction, which we attributed to inappropriate use of the
underlined AG to mark the end of the intron during splicing. This
defect was corrected by site-directed mutagenesis using the QuickChange
kit from Stratagene. The corrected cDNA coded for a full-length
TIA-1 protein. The chicken TIAR EST sequence identified
(dkfz426_14A13R1) appeared to start within the intron upstream of exon
6, but also contained exon 6-11 sequences. Missing 5' sequences were
obtained by carrying out RT-PCR on DT40 RNA using a downstream exon 8 primer (5'-gttagcccagaagcaat-3') derived from the EST sequence and an
upstream primer (5'-atggaagacgacgggcagcc-3') identical to the human
TIAR cDNA sequences coding for the first amino acids of human TIAR.
Two amplified fragments were obtained, subcloned, and sequenced. They
differed by the presence or absence of 51 base pairs, reflecting the
use of two alternative 3'-splice sites for exon 3. Missing 3'-sequences
were obtained from a composite sequence (044907.3), which became
available subsequently, and which was derived by combining several ESTs
(data from the UMIST/Nottingham/Dundee universities chicken EST
sequencing project, chick.umist.ac.uk/). Our final composite chicken
tiar cDNA sequences code for proteins of 388 and 371 amino acids. tia-1 and tiar probes were used to screen a chicken cosmid genomic library (library 125 from the Resource
Center of the German Human Genome Project Max-Planck-Institute for
Molecular Genetics, Berlin), using instructions supplied by the library
provider. Cosmid DNA was prepared from positive colonies. Maps of the
chicken tia-1 and tiar genes were made using
cosmid DNA by determining the sizes of PCR products obtained using
primers within adjacent exons for amplification across the intervening intron. Sequences determined here (chicken TIA-1 and TIAR
cDNA sequences, and the chicken tiar gene sequences
between exons 5 and 6 (including exons 5A and 5B), and between exons 10 and 11 (including exon 11A)) are available under GenBankTM
accession numbers AY189903-AY189906. Human TIA-1 and TIAR cDNA sequences are available under GenBankTM accession
numbers M77142 and M96954, respectively. The human tiar gene
sequence is available as EnsEMBL gene ENSG00000151923 at
www.ensembl.org.
Targeting Vectors--
Various drug resistance cassettes under
control of the chicken DT40 Cell Electroporation--
DT40 cells were kindly provided
by Jean-Marie Buerstedde and were maintained at 37 °C and 5%
CO2, in RPMI 1640 (Sigma) containing 10% fetal calf serum,
1% chicken serum (Sigma), 2 mM L-glutamine (Sigma), and 100 µM Growth Tests--
Different cell lines were first cultivated for
48 h in the absence of any antibiotic selection. They were then
seeded at a density of 3 × 105 cells/ml in
antibiotic-free medium containing 1% chicken serum and either 0, 1, or
10% fetal calf serum. Aliquots were removed at different times, and
cells counted. Viable and non-viable cells were distinguished by eosin staining.
DNA and RNA Analysis--
Isolation of DNA and RNA and Southern
blotting were carried out using standard techniques (33). RT-PCR was
carried out as described elsewhere (34, 35). Primers used were
5'-ggaagacgacgggcagcc-3' from tiar exon 1, with
5'-gcaaaggctgacttgata-3' from tiar exon 5;
5'-caatcacttccacgtgttcg-3' from tiar exon 5, with
5'-gttagcccagaagcaat-3' from tiar exon 8;
5'-gtactcatgggaggccag-3' from tiar exon 7 with 5'-aggctgagcaccaaatccacccat-3' from tiar exon 12. 24-28
cycles of amplification (1' at 94 °C; 1' at 50 °C; 2'30 at
72 °C) were used so as to remain in the range of exponential
amplification. PCR products were migrated on 2% agarose gels,
transferred to nylon filters (Hybond N+, Amersham Biosciences), and
hybridized with a 32P-labeled probe corresponding to
tiar exons 1-12 from the tiar cDNA.
Quantative PCR was done using the Brilliant Quantitative PCR Core
Reagent kit from Stratagene with cDNA from different lines, using
conditions specified by the manufacturer. Reactions were carried out
and analyzed using an MX-4000 apparatus from Stratagene. In this
technique an intercalating dye was used to quantify PCR products. For
each sample, two amplifications are carried out in parallel, one to
quantify tiar products, and one to quantify chicken actin
products, the latter quantification permitting comparison of different
samples. Primers used were in tiar exon 5 (5'-ccacgtgttcgttggggatttaag-3') and tiar exon 5A
(5'-gcacagttccttgcagttagcc-3') for quantification of exon 5A-containing RNA. For quantification of tiar RNA without any alternative
exon spliced between exons 5 and 6, a primer from tiar exon
5 (5'-cttccacgtgttcgttggg-3') and a primer crossing the tiar
exon 5/6 junction (5'-ccgtgcatccgatattttacc-3') were used. Actin
primers were 5'-tgggcttcatcaccaacgt-3' and 5'-gccccagacatcagggtgt-3'. Each primer pair yielded a single PCR product. Where indicated, cells
were incubated in cycloheximide (50 µg/ml) for 2 h before harvesting for RNA preparation.
Western Blotting--
Cells were lysed by repeated cycles of
freezing and thawing. Protein content of extracts was determined using
the BCA-200 protein assay kit from Pierce with bovine serum albumin as
a standard. Aliquots of protein (30 µg) were subjected to 10%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and incubated with goat polyclonal antibodies against TIA-1 or TIAR
(Santa Cruz Biotechnology, Inc.), or mouse antibodies against ASF/SF2
(gift from James Stévenin). Detection was carried out with a POD
kit from Roche Diagnostics.
Chicken TIA-1 and TIAR--
DT40 cells express a 40-kDa TIA-1
singlet comigrating with the faster moving band of the human TIA-1
doublet (data not shown). This doublet results from alternative
splicing of tia-1 exon 5 (36), so this suggests that DT40
TIA-1 mRNA does not contain an exon equivalent to human exon 5. A
TIA-1 cDNA identified in a chicken bursal EST data base (32) codes
for a 372 amino acid protein 95% identical to the human TIA-1 form
lacking exon 5-encoded sequences (see "Materials and Methods").
DT40 cell TIAR was detected as a 40-kDa doublet comigrating with the
human TIAR doublet (data not shown). Both doublets arise from use of
two alternative 3'-splice sites for tiar exon 3 (3).2 The amino acid sequence
of chicken TIAR was deduced from sequences of cDNA fragments we
amplified using RT-PCR on DT40 RNA (see "Materials and Methods").
The longer chicken TIAR isoform of 388 amino acids is 96% identical to
the corresponding isoform of human TIAR.
Amplified TIA-1 and TIAR cDNA fragments were used to screen a
chicken cosmid library, and several tia-1 and
tiar cosmid clones were isolated. These were used to
establish a map of the corresponding genes (Fig.
1A; note that exons are
numbered following the assumption that there is no chicken equivalent
of the alternatively spliced human tia-1 exon 5). A
schematic representation of chicken TIA-1 and TIAR is shown in Fig.
1B. These proteins are composed of three RRMs and a
glutamine-rich domain. In the human proteins, the second RRM is
required for (and indeed responsible for) their specific binding to
uridylate-rich sequences (19). Given the very high similarity observed
between the chicken and human proteins, it seemed safe to assume that
the second RRMs in the chicken proteins play the same roles. These RRMs
are encoded by exons 5 and 6 (note that we assume that there is no
chicken counterpart to the human alternative exon 5, so chicken
tia-1 exons 5 and 6 correspond to human tia-1
exons 6 and 7). We decided to disrupt the tia-1 and
tiar genes by insertion of sequences in exon 5, so as to
disrupt the sequences coding for RRM2, as shown in Fig. 1B
(see arrow insert).
tia-1 Gene Disruption in DT40 Cells--
One of the
tia-1 cosmid clones was used for construction of the
targeting vectors shown in Fig.
2A, in which either a neomycin resistance gene or a puromycin resistance gene has been inserted in
tia-1 exon 5. DT40 cells were electroporated with the
neomycin resistance targeting vector. DNA was isolated from 19 neomycin-resistant clones, digested by EcoRV, and analyzed
by Southern blotting with a tia-1 probe covering exons
6-10. This probe detects a 5.5-kb fragment in DT40 cell DNA (intact
allele in Fig. 2, A and B, lane 1) and
should detect an additional 7.8-kb fragment in clones with one
disrupted allele (Fig. 2A). This latter result was observed for four clones (see Fig. 2B, lanes 2 and
3 for two examples of such tia-1(+/ tiar Gene Disruption in DT40 Cells--
We made a tiar
targeting vector (Fig. 3A) in
which exon 5 was disrupted by insertion of a hygromycin B-resistance
gene. DT40 cells were electroporated with this vector. DNA from 19 hygromycin B-resistant clones was digested by EcoRV and
analyzed by Southern blotting with a tiar probe covering the
genomic region between exons 3 and 5. In DT40 cell DNA, the two
tiar alleles are detected on separate EcoRV
fragments of 12 and 7.6 kb (intact alleles 1 and 2 in Fig. 3,
A and B, lane 1). Successful targeting
of one of these alleles should lead to complete loss of one of these EcoRV fragments. This was observed for one of the 19 hygromycin B-resistant clones analyzed (Fig. 3B, lane
2). This clone was termed a tiar(1+/2
When tiar targeting vector 1 was electroporated into
tia-1(
We attempted without success to disrupt the remaining intact
tiar allele in clone 3-1 using a targeting vector similar to that shown in Fig. 3A, but containing a mycophenolic
acid-resistance or a histidinol resistance cassette in place of the
hygromycin B resistance cassette (a total of 26 histidinol-resistant
clones and 39 mycophenolic acid-resistant clones were analyzed). While this result is consistent with a requirement for TIA-1 or TIAR for cell
viability, no conclusion can be drawn from it, as the targeting
efficiency of the tiar vectors used is not very high (see
previous two paragraphs).
A Second tiar Targeting Vector--
We tried to improve targeting
efficiency using a vector with a mycophenolic acid-resistance cassette
(gpt sequence) designed to target a different region of the
tiar gene. Targeting vector 2, and the expected results of
succesful tiar gene disruption using it, are shown in Fig.
4A. In any allele disrupted by
recombination with this vector, a 3'-splice site followed by two
polyadenylation signals (pA) will be found in the intron following exon
2. The disrupted allele can only yield a truncated mRNA containing
exons 1 and 2, followed by part of intron 2, and so will be incapable of furnishing functional TIAR. Note that in clones 3-1 and 3-2, the
vector can target either the intact tiar allele (to generate a gpt-disrupted allele), or the allele already disrupted by insertion of the hygromycin B-resistance gene (to generate a hygro+gpt-disrupted allele).
Targeting vector 2 was electroporated into clones 3-1 and 3-2, and DNA
from mycophenolic acid-resistant clones was subjected to
EcoRV digestion and analysis by Southern blotting with a
probe corresponding to exon 2 and 2 kb of downstream intron sequences. For the clone 3-2 electroporation, we analyzed 30 clones. The intact
tiar allele 1 was disrupted in none of these clones.
However, in 6 of them the hygro-disrupted allele 2 had been targeted a second time, to yield cells with an intact allele 1 and a
hygro+gpt-disrupted allele 2 (3-2* cells, see Fig. 4B,
lane 2 for an example). For the clone 3-1 electroporation,
we analyzed 55 clones. Once again, the intact tiar allele 2 was disrupted in none of these clones, but in 9 of them the already
hygro-disrupted allele 1 had been targeted a second time, to yield
cells with an intact allele 2 and a hygro+gpt-disrupted allele 1 (3-1*
cells, see Fig. 4C, lane 2 for an example).
Both tiar Alleles Can Be Disrupted Only in Cells Expressing
TIA-1--
With targeting vector 2, only clone 3-1 and 3-2 cells in
which an already disrupted tiar allele has been targeted a
second time were obtained. This strongly suggests that cells expressing neither TIA-1 nor TIAR are not viable. However, we could not
rule out that the already disrupted allele was much more susceptible to
targeting than the intact allele for some reason. We felt that it might
be possible to disrupt the remaining intact tiar allele if
TIA-1 expression was first restored. We prepared clone 3-1 cells
re-expressing TIA-1 as a result of stable transfection with an
epitope-tagged TIA-1 expression vector (clone 3-1+TIA-1 cells). These
cells express levels of TIA-1 significantly higher than those expressed
by DT40 cells (Fig. 2C, compare lanes 5 and
6 to lane 1). The cells were electroporated with
targeting vector 2 (Fig. 4A), and mycophenolic
acid-resistant clones were selected. In 4 of 46 clones analyzed, the
already disrupted tiar allele 1 had been targeted a second
time (data not shown). However, in 4 other clones the intact allele 2 had been targeted, to yield cells with no intact tia-1
allele and no intact tiar allele (clone 4+TIA-1 cells, see
Fig. 4C, lane 3 for an example). These results demonstrate that the intact allele is as susceptible to targeting as
the already disrupted allele. Clone 4+TIA-1 cells do not contain TIAR
(Fig. 4D, lane 5; the very weak band at 40 kDa is
due to cross-reaction of the antibodies used with the epitope-tagged TIA-1).
In summary, electroporation of targeting vector 2 into cells with both
tia-1 alleles and one tiar allele disrupted led
to isolation of 15 clones with the already disrupted allele targeted a
second time, but no clones with the intact tiar allele
targeted, despite the fact that intact and disrupted tiar
alleles are equally susceptible to targeting. However,
tia-1( Reduced Growth and Survival of tia-1( TIA-1 Overexpression Prevents TIAR Expression by Inducing Splicing
of Exons with Stop Codons--
While analyzing the expression of TIAR
in clone 3-1+TIA-1-derived cells, we noticed that clone 3-1+TIA-1 does
not express TIAR (Fig. 4D, compare lanes 2 and
3), despite the fact that it contains one intact
tiar allele. Clone 3-1+TIA-1 overexpresses TIA-1 as compared
with DT40 cells (Fig. 4D, compare lanes 1 and 3). We also analyzed a clone 3-1+TIA-1 derivative (clone
3-1*+TIA-1, see above), in which the already targeted tiar
allele 1 has been targeted a second time, but which still contains one
intact tiar allele. In this derivative, TIA-1 levels had
dropped to levels similar to those seen in DT40 cells, and TIAR
expression was restored (Fig. 4D, lane 4). These
results suggest that TIA-1 overexpression prevents TIAR expression.
We have shown previously that TIA-1 overexpression induces the splicing
of alternative human tiar exons with premature stop codons
(21), although we did not investigate the effect of this on TIAR
levels. We wondered if this mechanism was conserved in chicken cells,
and so studied TIAR mRNA expression patterns in clones 3-1 and
3-1+TIA-1 by an RT-PCR approach. Using primers in exons 1 and 5, little
difference was observed between the two clones (Fig.
6A, compare lanes 1 and 2; two fragments were obtained as there are two
alternative 3'-splice sites for exon 3). However, we detected some
RT-PCR products (products a, b, and c) present only in the clone
3-1+TIA-1 sample when we used a primer pair in exons 5 and 8, or a
primer pair in exons 7 and 12 (Fig. 6A, compare lanes
3 and 4, and lanes 5 and 6,
respectively). These products were not detected in RNA from clone
3-1*+TIA-1 (data not shown).
Note that the results shown for lane 4 result
from a 12-fold longer exposure of the film than that used for
lane 3. This is a reflection of the fact that TIAR mRNA
levels are much lower in clone 3-1+TIA-1 cells than in clone 3-1 cells
(as discussed below, and see Fig.
7C for quantification of this
difference), while RNAs with exons 5A and 5B included are unstable. The
new products (marked a, b, and c in
Fig. 6A) were cloned and sequenced, and their sequences
compared with the chicken TIAR cDNA sequence (data not shown), and
to selected parts of the chicken tiar gene. Comparison of
these sequences showed that product b is the result of splicing of two
additional exons (5A and 5B) between exons 5 and 6. In product a, the
intron between exons 5A and 5B is retained. Product c is the result of
splicing of an additional exon (10A) between exons 10 and 11. All these
additional exons contain in-frame stop codons (Fig. 6B), and
their splicing explains why clone 3-1+TIA-1 does not contain
detectable levels of normal TIAR. The 5'-splice sites of exons 5A and
5B are followed by a uridylate-rich sequence, as expected for
TIA-1-activated sites (Fig. 6B). This is not the case for
exon 10A, however.
TIA-1/TIAR Is Required for Exon 5A Splicing--
RT-PCR
analysis of RNA from DT40 cells with primers in tiar exons 5 and 8 shows that these cells contain very low levels of mRNAs with
exons 5A and 5B included (Fig. 7A, lane 1). This
could be because inclusion of these exons with their in-frame stop
codons exposes resulting mRNAs to nonsense-mediated mRNA decay
(37). To test this, we incubated DT40 cells in the presence of the
protein synthesis inhibitor cycloheximide to block nonsense-mediated
mRNA decay. This treatment lead to a significant increase in the
levels of TIAR mRNAs containing exon 5A spliced between exons 5 and
6, or both exon 5A and exon 5B spliced between exons 5 and 6 (Fig. 7A, compare lanes 1 and 2). This was
confirmed (Fig. 7B) by carrying out quantitative RT-PCR on
RNA from DT40 cells incubated or not in cycloheximide, using primers in
exons 5 and 5A as shown in Fig. 7B. Use of this primer pair
will lead to amplification of the same DNA fragment from RNA with exon
5A spliced between exons 5 and 6, and from RNA with both exons 5A and
5B spliced between exons 5 and 6. The primer pair can thus be used to
quantify total exon 5A splicing.
In contrast, cycloheximide treatment of DT40 cells did not induce any
significant increase in normal TIAR mRNA levels in DT40 cells or
clone 3-1 cells (Fig. 7C), as judged by quantitative RT-PCR
using a primer in exon 5 together with a primer crossing the exon
5-exon 6 junction of normal TIAR mRNA (this primer pair should
detect TIAR mRNA with exon 6 spliced to exon 5, but not mRNA
with exons 5A or 5B spliced to exon 5). Very low levels of normal TIAR
mRNA were detected in clone 3-1+TIA-1 cells (Fig. 7C).
This result is not surprising, as in these cells exons 5A or 5B are
spliced very efficiently.
To test for the effects of TIA-1/TIAR depletion on exon 5A splicing,
quantitative RT-PCR was carried out on RNA from various cell lines with
disrupted genes (incubated or not in cycloheximide) using primers in
exons 5 and 5A to detect exon 5A-containing mRNAs (Fig.
7B). Note that in Fig. 7B results shown represent
the levels of exon 5A-containing mRNAs obtained from one
tiar allele, i.e. we have corrected for the fact
that some of the cell lines tested have one tiar allele,
while others have two. As shown in Fig. 7B, TIA-1 depletion
leads to a drop in exon 5A splicing (tia-1(
What is the magnitude of the effect that fluctuations of exon 5A
splicing can have on TIAR protein levels? It should be noted that in
DT40 cells, about 30% of TIAR mRNAs contain exon 5A (Fig. 7A, lane 2). So if no exon 5A splicing at all
were to take place in DT40 cells for some reason, TIAR mRNA and
protein levels would be expected to increase by a maximum of about
1.4-fold. A lower increase would be observed if any additional
mechanism (based for example on translational control) exists to limit
fluctuations in TIAR levels.
Exons 5A and 5B and Parts of Flanking Introns Are Almost Identical
in the Chicken and Human tiar Genes--
The chicken exons 5A and 5B
are identical in sequence to a corresponding pair of TIA-1-activated
exons from the human tiar gene (see Fig. 3 in Ref. 21; note
that the numbering system used for human tiar exons in Ref.
21 differs from that used for chicken tiar exons here, and
so human tiar exons 6 and 7 in Ref. 21 correspond in fact to
chicken tiar exons 5 and 6). This high level of sequence
conservation extended to the intron separating exons 5A and 5B, and to
part of the intron sequences flanking these exons (Fig.
8A). The strong conservation
of exons 5A and 5B and flanking intron sequences between chickens and
humans suggests that their splicing is physiologically important.
Although the intron between human tiar exons 11 and 12 (which correspond to chicken tiar exons 10 and 11) contains
a TIA-1-activated exon, exon 11A (21), this exon shows no significant
similarity to chicken exon 10A, nor do any other portions of the human
and chicken introns (Fig. 8B).
We have isolated DT40 cells
(tia-1( The results resumed above indicate that TIA-1 and TIAR can play
redundant roles in cell viability. Consistent with this, while the
serum dependence for growth and survival of tia-1( Depletion of TIA-1/TIAR leads to cell death, but so does overexpression
of these proteins. Although the effects of changing TIA-1/TIAR levels
on cell viability could be the consequence of problems with either
translational control or splicing control, an explanation based on
splicing control is particularly easy to envisage. Furthermore, such an
explanation would be consistent with observations that several genes
involved in apoptosis can code for both pro-apoptotic and
anti-apoptotic proteins, depending on the alternative splicing choice
made when splicing their pre-mRNAs (38). Clearly TIA-1/TIAR
depletion could significantly reduce splicing of some exons (as
observed here for tiar exon 5A), and this could lead to cell
death (see exon 2 in Fig. 9). On the
other hand, TIA-1/TIAR overexpression could increase splicing of some other exons (as it does for tiar exons 5A and 5B), and this
could also lead to cell death (see exon B in Fig. 9).
/
)tiar(
/+) cells)
are viable. However, their growth and survival in medium containing low
serum levels is significantly reduced compared with DT40 cells. The
remaining intact tiar allele in
tia-1(
/
)tiar(
/+) cells can only be
disrupted if TIA-1 expression is first restored to the cells by
transfection of a TIA-1 expression vector. We conclude that DT40 cells
require either TIA-1 or TIAR for viability. TIA-1 overexpression in
tia-1(
/
)tiar(
/+) cells leads to a radical drop in TIAR levels, by inducing efficient splicing of two
tiar alternative exons carrying in-frame stop codons. In
wild-type DT40 cells, tiar transcripts including these
exons can also be detected. These transcripts increase significantly in
abundance in cycloheximide-treated cells, suggesting that splicing of
the exons exposes mRNAs to nonsense-mediated mRNA decay. TIA-1
or TIAR depletion leads to a marked drop in splicing of the
exons. The human tiar gene contains a corresponding
pair of TIA-1-inducible alternative exons, and we show that there is
very high sequence conservation between chickens and humans of the exon
pair and parts of the flanking introns. The TIA-1/TIAR responsiveness
of these alternative tiar exons is likely to be of
physiological importance for controlling TIAR levels.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) mice die between embryonic day 16.5 and 3 weeks
of age, though surviving mice appear normal (7). Mice with disrupted
tiar genes show high levels of embryonic lethality, from 90 to 100% depending on the strain analyzed (7, 26). Mice with both
tia-1 and tiar genes disrupted die before
embryonic day 7 (7). These results may be a sign that TIA-1 or TIAR are required for some specific process (possibly involving apoptosis) required for proper development, which, if not carried out, leads to
early embryonic lethality. The results could thus be in agreement with
the proposed apoptosis-promoting activity for TIA-1 and TIAR. Alternatively, they may indicate that the proteins are in fact necessary for cell viability. It was important to distinguish between
these two possibilities.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin promoter and linked to either an SV40
or a thymidine kinase gene polyadenylation signal were kindly provided
by Jean-Marie Buerstedde. Targeting vectors as shown in Figs. 2-4 were
made by standard subcloning procedures (33). The tia-1 gene
targeting vectors contain either a 2.3-kb neomycin resistance cassette, or a 2.3-kb puromycin resistance cassette, flanked by tia-1
gene sequences (4.1-kb upstream and 4.9-kb downstream). tiar
gene-targeting vector 1 contains a hygromycin B resistance cassette,
flanked by tiar gene sequences (4.0-kb upstream and 4.3-kb
downstream). tiar gene-targeting vector 2 contains a
mycophenolic acid resistance cassette (gpt sequence). The gpt sequence
is preceded by a 3'-splice site and polyadenylation sequence taken from
pCI-neo (Promega). This composite block is flanked by tiar
gene sequences (2-kb upstream and 2-kb downstream).
-mercaptoethanol (Sigma). For each
transfection, 107 cells were washed and resuspended in 800 µl of phosphate-buffered saline containing 30 µg of linearized
plasmid, before electroporation using a Gene Pulser apparatus (Bio-Rad)
set to 550 V and 25 µF. After electroporation, cells were added to 20 ml of fresh culture medium and 100-µl aliquots transferred to each
well of a 96-well plate. After 24 h, 100 µl of fresh medium
containing the appropriate antibiotic was added. After 7-10 days,
resistant cells were transferred to wells of a 24-well plate, and
subsequently further expanded. The following final concentrations of
antibiotics were used: G418, 2 mg/ml; puromycin, 0.5 µg/ml;
hygromycin B, 1 mg/ml; histidinol, 1 mg/ml; mycophenolic acid, 25 µg/ml; added together with xanthine to 250 µg/ml and hypoxanthine
to 15 µg/ml. All antibiotics were from Sigma, except for G418, which
was from Invitrogen.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representations of tia-1
and tiar genes and proteins. A,
the exon-intron structure of the genes is shown, with exons marked as
black boxes and introns as lines. We have not
determined the size of the intron between tiar exons 1 and
2, and so this intron is marked by a broken line.
B, the structures of TIA-1 and TIAR are shown, with their
three RNA recognition motifs (RRM-1 to 3) and glutamine-rich domains
(Q-Rich). For RRM-2, the two RNP motifs are represented by
black boxes. The parts of TIA-1 and TIAR encoded by exons 5 and 6 (ex 5 and ex 6) are shown, as is the
position within exon 5 where drug resistance markers were inserted
(inset).
) clones).
One of the tia-1(+/
) clones was subsequently electroporated with the puromycin resistance targeting vector. Out of
29 puromycin-resistant clones analyzed as above, four appeared to have
both tia-1 alleles disrupted; the 5.5-kb fragment
representing an intact allele had disappeared, and only 7.8-kb
fragments representing disrupted alleles were detected (see Fig.
2B, lanes 4 and 5 for two examples).
Western blotting with antibodies against TIA-1 showed that these
tia-1(
/
) clones no longer produce TIA-1 (Fig. 2C, lanes 3 and 4; the very weak
doublet observed at 40 kDa is due to slight cross-reactivity of the
antibodies with TIAR).
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Fig. 2.
tia-1 disruption.
A, schematic representations of the tia-1
targeting vector, an intact tia-1 allele, and the disrupted
tia-1 allele obtained by targeting of the intact allele.
Drawings are not to scale. Exons are represented by numbered
boxes. Neo/puro represent 2.3-kb DNA fragments corresponding to
neomycin or puromycin resistance cassettes. The origins of
EcoRV fragments visualized in B are shown.
B, Southern blot. DNA from cells as indicated was digested
by EcoRV, migrated on an agarose gel, and transferred to a
nylon filter before hybridization with a probe covering
tia-1 exons 6-10 derived from the TIA-1 cDNA.
tia-1(+/ ), cells with one tia-1 allele
disrupted. tia-1(
/
), cells with both tia-1
alleles disrupted. C, Western blot. Proteins from cells as
indicated (clone 3-1+TIA-1 is a clone with both tia-1
alleles and one tiar allele disrupted, and which expresses
TIA-1 from an integrated expression vector) were migrated on a 10%
SDS-polyacrylamide gel before transfer to a nitrocellulose filter and
analysis with antibodies against TIA-1. Protein samples were also
analyzed with antibodies against ASF/SF2 as a control.
) clone to
indicate that allele 2 has been disrupted. Both tia-1
alleles were disrupted in this clone by successive use of the two
tia-1 targeting vectors shown in Fig. 2A. The
resulting clone was named clone 3-2 (Fig. 3B, lane
3), as it contains three inactivated alleles: two tia-1
alleles and tiar allele 2.
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Fig. 3.
Disruption of a tiar allele
with targeting vector 1. A, schematic representations
of the tiar targeting vector, intact tiar alleles
1 and 2, and disrupted tiar alleles obtained by targeting of
the intact alleles. Drawings are not to scale. Exons are represented by
numbered boxes. Hygro represents a 3.3-kb DNA fragment
corresponding to a hygromycin B resistance cassette. The origins of
EcoRV fragments visualized in B are shown. In
clone 3-1 tiar allele 1 is disrupted, and in clone 3-2 tiar allele 2 is disrupted as shown. B, Southern
blot. DNA from cells as indicated was digested by EcoRV,
migrated on an agarose gel and transferred to a nylon filter before
hybridization with a probe covering tiar exons 3-5 derived
from a tiar cosmid. tiar(1+/2 ), cells with
tiar allele 2 disrupted. Clone 3-1, cells with both
tia-1 alleles and tiar allele 1 disrupted. Clone
3-2, cells with both tia-1 alleles and tiar
allele 2 disrupted. The weak band marked by an asterisk in
lane 1 is an overdigestion product. C, Western
blot. Proteins from cells as indicated were migrated on a 10%
SDS-polyacrylamide gel before transfer to a nitrocellulose filter and
analysis with antibodies against TIAR. Protein samples were also
analyzed with antibodies against ASF/SF2 as a control.
/
) cells, a tiar allele (allele 1 this
time) was inactivated in one of the 19 hygromycin B-resistant clones
tested (Fig. 3B, lane 4). This clone was named
clone 3-1, as it contains three inactivated alleles: two
tia-1 alleles and tiar allele 1. Note that a
different tiar allele is disrupted in clones 3-1 and 3-2. All of the clones discussed above contain at least one intact tiar allele and continue to express TIAR as a doublet (Fig.
3C).
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Fig. 4.
tiar allele disruption with
targeting vector 2. A, schematic representations of
tiar targeting vector 2, intact tiar alleles 1 and 2, hygro-disrupted alleles 1 and 2 as in Fig. 3, and disrupted
tiar alleles obtained following targeting by vector 2 of the
intact alleles or the hygro-disrupted alleles. Drawings are not to
scale. Exons are represented by numbered boxes. hygro
represents a 3.3-kb DNA fragment corresponding to a hygromycin B
resistance cassette, while gpt represents a mycophenolic acid
resistance cassette. 3'-ss and pA are a 3'-splice site sequence and a
polyadenylation signal, respectively. The origins of EcoRV
fragments visualized in B are shown. B and
C, Southern blots. DNA from cells as indicated was digested
by EcoRV, migrated on an agarose gel, and transferred to a
nylon filter before hybridization with a probe covering tiar
exon 2 and 2 kb of downstream intron. Clone 3-1 and clone 3-2 are as in
the legend to Fig. 3. Clone 3-1*, clone 3-1 cells with the
hygro-disrupted allele 1 targeted a second time. Clone 3-2*, clone 3-2
cells with the hygro-disrupted allele 2 targeted a second time. Clone
4+TIA-1, clone 3-1 cells with both tia-1 and both
tiar alleles disrupted, but which express TIA-1 from an
integrated expression vector. The clone 4+TIA-1 sample was run on the
same gel as the two other samples, but not on an adjacent lane on the
gel. The three samples have been regrouped to save space here.
D, Western blot. Proteins from cells as indicated were
migrated on a 10% SDS-polyacrylamide gel before transfer to a
nitrocellulose filter and analysis with antibodies against TIAR
or TIA-1. Clone 3-1+TIA-1 and clone 3-1*+TIA-1 are respectively clone
3-1 and clone 3-1* cells that express TIA-1 from an integrated
expression vector. For the TIA-1 blot, all samples were run on the same
gel, but samples for lanes 1-3 and lanes 4-5
were not on adjacent lanes on the gel. They have been regrouped to save
space here.
/
)tiar(
/
) cells expressing TIA-1
from an integrated expression vector can be obtained readily. This is a
very strong indication that
tia-1(
/
)tiar(
/
) cells are not viable, and
shows in addition that TIA-1 can functionally replace TIAR for
viability. As tia-1(
/
) cells are viable, TIAR can also
replace TIA-1 for viability.
/
)tiar(
/+) Cells in Low
Serum--
As tia-1(
/
)tiar(
/
) cells are
not viable, we wondered if the viable
tia-1(
/
)tiar(
/+) cells (clones 3-1 and 3-2)
had more stringent requirements for growth or survival than DT40 cells. We tested growth of various lines in medium containing 0, 1, or 10%
fetal calf serum (and 1% chicken serum). The results of a typical
series of experiments are shown in Fig.
5. There is no significant difference in
growth between DT40 cells, tia-1(
/
) cells,
tiar (+/
) cells, clone 3-1 cells, and clone 3-2 cells in
medium containing 10% fetal calf serum (Fig. 5, A and
B). In medium containing 1 or 0% fetal calf serum there is
significant mortality of DT40 cells, tia-1(
/
) cells and
tiar(+/
) cells, but these cells continue to proliferate,
and the net result is an increase in the number of viable cells with
time (Fig. 5A). However, for clones 3-1 and 3-2, the number
of viable cells remained almost stationary in medium containing 1%
fetal calf serum, and actually dropped rapidly in medium containing 0%
fetal calf serum (Fig. 5B), with a concomitant increase in
the number of dead cells observed (data not shown). This suggests that
the tia-1(
/
)tiar(
/+) cells are unable to
survive under these reduced serum conditions. This defect was however
eliminated (Fig. 5B) when
tia-1(
/
)tiar(
/+) cells were forced to
re-express TIA-1 from an expression vector (we used the clone 3-1+TIA-1
cells described above). Clone 4+TIA-1 cells behaved like DT40 cells
under all the conditions tested (data not shown).
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Fig. 5.
Growth curves. Cells were inoculated
into medium containing 1% chicken serum, and 0, 1, or 10% fetal calf
serum as shown, and the viable cell count determined after various
times of incubation. Each experiment was carried out at least three
times, and typical results are shown.
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Fig. 6.
TIA-1 overexpression leads to splicing of
tiar alternative exons. A, RNA from
clone 3-1 and clone 3-1 expressing TIA-1 from an integrated expression
vector (clone 3-1+TIA-1) was used for RT-PCR analysis with primers in
tiar exons 1 and 5 (lanes 1 and 2), or
in exons 5 and 8 (lanes 3 and 4), or in exons 7 and 12 (lanes 5 and 6). PCR products were
migrated on an agarose gel and transferred to a nylon filter before
hybridization with a tiar cDNA probe. Bands marked
a, b, and c appearing in clone
3-1+TIA-1 only are identified, and their structures are represented
schematically. Note that the results shown for lane 4 result
from a 12-fold longer exposure of the film than that used for the other
lanes. B, sequences of alternative tiar exons 5A,
5B, and 10A activated by TIA-1. Exon sequences are marked in
capital letters. Stop codons in the reading frame of the
upstream tiar exon are marked by asterisks.
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Fig. 7.
tiar alternative exon splicing
drops when TIA-1/TIAR is depleted. A, RNA from cells as
marked that had been incubated alone or in the presence of
cycloheximide (50 µg/ml) for 2 h was used for RT-PCR analysis
using primers in tiar exons 5 and 8. PCR products were
migrated on an agarose gel and transferred to a nylon filter before
hybridization with a tiar cDNA probe. Bands
corresponding to splicing of exon 5A alone, or exon 5A and 5B together
are identified (their identities were established by sequencing).
B and C, quantitative PCR. Each bar
represents the mean with S.D. of the -fold increase of the sample over
that of the DT40 sample. Samples of cDNA from cells as shown were
subjected to quantitative PCR using for B primers in exons 5 and 5A so
as to quantify together cDNAs with either exon 5A or exon 5A
spliced to exon 5B or for C a primer in exon 5 and a primer crossing
the exon 5/exon 6 junction, to quantify cDNAs corresponding to
normal TIAR mRNA without exon 5A or exon 5B. For B,
results were compared with the level of exon 5A splicing in DT40 cells
not exposed to cycloheximide, to which the value 1 was arbitrarily
attributed. For B, results shown represent the levels of
exon 5A-containing mRNAs or TIAR mRNAs obtained from one
tiar allele, i.e. we have corrected for the fact
that some lines have one tiar allele, while others have two.
For C, results were compared with the level of exon 5 to
exon 6 splicing in DT40 cells not exposed to cycloheximide, to which
the value 1 was arbitrarily attributed. Determinations were repeated
four times, and mean values are shown.
/
) cells). A
more marked drop is observed in tiar(+/
) cells lacking one
tiar allele. In cells lacking both tia-1 alleles
and one tiar allele (clones 3-1 and 3-2), splicing of exon
5A falls to very low levels (see also Fig. 7A, compare
lanes 3 and 4 for clone 3-1 and lanes
5 and 6 for clone 3-2). Exon 5A splicing is thus truly
responsive to TIA-1/TIAR levels, decreasing when levels fall, and
increasing when levels rise.
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Fig. 8.
Comparison of human and chicken
tiar genes. A, comparison of the human
tiar exon 6-7 region containing the TIA-1-activated
alternative 6A and 6B exons, to the corresponding region of the chicken
gene containing the TIA-1-activated alternative 5A and 5B exons. Note
that the human exons 6 and 7 correspond to the chicken exons 5 and 6. Regions where the sequence homology is greater than 95% are shown by
heavy black lines. B, comparison of the human
tiar exon 11-12 region containing the TIA-1-activated
alternative 11A exon, to the corresponding region of the chicken gene
containing the TIA-1-activated alternative 10A exon. Note that the
human exons 11 and 12 correspond to the chicken exons 10 and 11. Regions where the sequence homology is greater than 85% are shown by
heavy black lines.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
)tiar(+/
) cells) with both
tia-1 alleles and one tiar allele disrupted. We
have not been able to disrupt the intact tiar allele in
these cells, our attempts to do so all leading to retargeting of the
already disrupted allele. However, if
tia-1(
/
)tiar(+/
) cells are
forced to express TIA-1 by transfection of a cDNA
expression vector, targeted disruption of their remaining intact
tiar allele then becomes possible. We conclude that DT40
cells require either TIA-1 or TIAR for viability. This suggests that
the embryonic lethality observed following disruption of
tia-1 or tiar genes in mice (7, 26) is due to
reduced cell viability, rather than problems with some specific process
required for proper development.
/
) and tiar(+/
) cells is indistinguishable from that of DT40
cells, it is markedly higher in cells obtained from
tia-1(
/
) cells by inactivating a tiar allele,
or in cells obtained from tiar(+/
) cells by inactivating
both tia-1 alleles. However, TIA-1 could be a less effective
mediator of tasks required for cell viability than TIAR. The
tia-1(
/
)tiar(+/
) cells expressing TIA-1
from an expression vector that were used for disruption of both
tiar alleles contain more TIA-1 than DT40 cells, and it may
be that disruption of both tiar alleles is only possible in
DT40 cells expressing higher than normal levels of TIA-1. Thus despite
our best efforts we have not been able to disrupt both tiar
alleles in DT40 cells.2 While it is possible to obtain
mouse cells lacking TIAR (7), the cells obtained express more TIA-1
than do wild-type cells (10). The notion that TIA-1 is less effective
than TIAR is also consistent with the lower embryonic lethality
observed with mice lacking TIA-1 as compared with mice lacking TIAR (7,
26).
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Fig. 9.
Hypothesis for the effect of changing
TIA-1/TIAR levels on cell viability. For the gene with exons 1, 2, and 3, splicing of exon 2 is required for viability. Depletion of
TIA-1/TIAR prevents splicing of this exon, and leads to cell death. For
the gene with exons A, B, and C, splicing of exon A to exon C is
required for viability. Overexpression of TIA-1/TIAR activates splicing
of exon B, and leads to cell death.
So should TIA-1 and TIAR be looked upon as apoptosis-promoting proteins (17, 20), or as proteins required for cell survival, which are inactivated during apoptosis? Upon Fas induction of apoptosis, TIA-1 becomes phosphorylated (24), and TIAR is relocated to the cytoplasm (25). It could be that TIA-1 is activated by phosphorylation for participation in events necessary for apoptosis progression. On the other hand, it is possible that TIA-1 phosphorylation is one facet of a general mechanism for splicing inhibition during apoptosis by inactivation of splicing factors (39-41). Similarly, TIAR translocation to the cytoplasm could actively promote apoptosis by bringing it into contact with cytoplasmic substrates (25), but it is equally possible that reducing nuclear TIAR levels is another facet of this general mechanism for splicing inhibition. The observations that overexpression of TIA-1 or TIAR can lead to apoptosis are difficult to interpret, as there is no evidence that such overexpression occurs normally. In addition, it is possible that overexpression of many splicing factors necessary for cell viability (such as ASF/SF2, for example (42)) will perturb splicing sufficiently to induce apoptosis. Interestingly, it has recently been shown that recombinant Sendai viruses expressing a short transcript that binds TIAR are non-cytopathic (23). Apoptosis triggered by 20-hydroxyecdysone during pupal metamorphosis of the silkworm Bombyx mori is accompanied by an increase in TIAR mRNA levels (43). Ischemia-induced apoptosis also leads to an increase in TIAR mRNA levels (44, 45). These observations suggest that TIAR could be required for apoptosis. Maybe TIA-1 and TIAR normally work for cell survival, but during apoptosis they are modified, so as to be rendered inactive for cell survival tasks, yet active to participate in new tasks in apoptosis progression. One possible way of achieving this could be by providing a new interacting partner for them.
All previous work implicating TIA-1/TIAR in splicing control has involved in vitro studies, or transient transfection of exogenous TIA-1/TIAR expression vectors or reporter genes (16, 17, 21). However, definitive evidence for the involvement of TIA-1/TIAR in splicing control required a demonstration that reducing the levels of endogenous TIA-1/TIAR alters the splicing of an endogenous pre-mRNA. The chicken lines with disrupted tia-1 and tiar genes have now allowed us to provide such evidence; reducing the levels of endogenous TIA-1/TIAR reduces the splicing of tiar alternative exon 5A on the endogenous tiar pre-mRNA. Splicing of the alternative 5A exon decreases in cells with both tia-1 alleles disrupted, and a greater decrease is seen in cells with one tiar allele inactivated. In cells with both tia-1 alleles and one tiar allele disrupted, splicing of the exon drops to very low levels. These results underscore the overlapping functions of TIA-1 and TIAR in splicing activation.
Exon 5A together with exon 5B make up a pair of chicken tiar alternative exons with in-frame stop codons whose splicing increases markedly following TIA-1 overexpression. The results reported here extend significantly our previous observation (21) that transient overexpression of TIA-1 activates splicing of alternative human tiar exons with stop codons, as we now show that stable TIA-1 overexpression blocks production of TIAR protein, and that this involves two phenomena. In addition to blocking TIAR production by introducing premature stop codons into the TIAR mRNA, inclusion of the alternative exons appears to expose mRNAs to nonsense-mediated mRNA decay (37); the abundance of mRNAs with the exons included rises significantly in cells treated with cycloheximide.
Negative feedback regulation of splicing as we have observed for the
tiar gene appears to be a common mechanism controlling the
level of splicing regulators, with for example the SR protein SC35
autoregulating its expression by promoting splicing events affecting
the 3'-UTR of its mRNAs and which expose them to nonsense-mediated mRNA decay (46). The extremely high sequence conservation between the human and chicken tiar genes of the stop
codon-containing alternative tiar exons, and indeed of parts
of the introns flanking them, is in favor of physiological importance
for the negative feedback regulation of tiar alternative
exon splicing. Clearly, TIA-1/TIAR-induced splicing of stop
codon-containing exons could be used to stop TIA-1/TIAR levels from
rising to high levels. This could be important, as high levels of these
proteins can induce apoptosis. On the other hand, if TIA-1/TIAR
levels drop below normal levels, this could be deleterious for cell
viability. However, splicing of stop codon-containing exons should fall
as a result, and this could lead to a rise in TIAR mRNA and protein levels. Finally, it is tempting to wonder if controlling splicing of
stop codon-containing exons is the only mechanism used to limit fluctuations of TIAR levels; could TIA-1 and TIAR, proteins known to be
involved in translational control (5-15), perhaps repress translation
of their own mRNAs?
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Cécile Bénézech for help with plasmid construction, and James Stévenin and Jean-Marie Buerstedde for kind gifts of materials.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Association pour la Recherche sur le Cancer and the Ligue Nationale contre le Cancer, Comité Departemental de Loire-Atlantique.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/EBI Data Bank with accession number(s) AY189903, AY189904, AY189905, and AY189906.
To whom correspondence should be addressed: INSERM U463, Institut
de Biologie-CHR, 9 Quai Moncousu, 44093 Nantes Cedex 1, France. Tel.:
33-0-2-40-08-47-50; Fax: 33-0-2-40-35-66-97; E-mail: breathna@nantes.inserm.fr.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M212378200
2 C. Le Guinet and R. Breathnach, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RRM, RNA recognition motif; EST, expressed sequence tag.
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REFERENCES |
---|
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