(Received for publication, March 14, 1995; and in revised form, July 31, 1995)
From the
Engagement of the T cell antigen receptor (TCR) causes transformed T cell hybridomas to produce lymphokines and then die by an apoptotic mechanism. Here we show that these functional effects of TCR-mediated signaling are associated with the accumulation of several cytosolic mRNA-binding proteins including the previously described AU-A, -B, and -C proteins as well as a novel 70-kDa protein. The results indicate that the 70-kDa protein derived from a 90-kDa precursor present in unactivated T cells and that both proteins bound to two independent sites at the 3`-end of the interleukin-2 mRNA, one in the coding region and the other in an AU-rich segment of the 3`-untranslated region. Glucocorticoids, TCR engagement by monoclonal antibodies, pharmocologic mimics of TCR signaling, or high concentrations of protein or RNA synthesis inhibitors all induced apoptosis, the cytosolic appearance of the RNA-binding proteins, and an increased rate of RNA turnover. Moreover, drugs that interfere with TCR-mediated signals, such as cyclosporin A and staurosporin, prevented both apoptosis and the appearance of the RNA-binding factors. The fact that the accumulation of these factors occurred in the presence of inhibitors of transcription and translation suggested that these proteins are present in an inactive form in unstimulated T cells and are activated when apoptosis is induced.
Several hormonal and developmental signals have been shown to
modulate gene expression by altering mRNA stability or mRNA
degradation(1) . Post-transcriptional regulation of mRNA
expression has been shown to play an important role in controlling the
level of rapidly induced mRNAs. This level of regulation is
particularly important for proteins that are active for brief periods,
such as growth factors, transcription factors, and proteins that
control cell cycle progression(1) . mRNA stability is
controlled by specific cis-acting elements including an AU-rich
sequence that is found in the 3`-untranslated region (UTR) ()of a variety of unstable mRNAs (2) and has been
shown to confer instability to otherwise stable RNAs(3) . The
AU-rich element (ARE) consists of multiple copies of the AUUUA
pentanucleotide and/or uridine-rich segments. These elements are
thought to target mRNA to rapid degradation by binding proteins
involved in RNA turnover(4) , and several AU-specific,
RNA-binding proteins, have been described
previously(5, 6, 7) . In human T cells,
Bohjanen et al.(8, 9) , reported that T cell
antigen receptor (TCR)-mediated signals induced three different
AU-specific RNA-binding proteins: AU-A (34 kDa), AU-B (30 kDa), and
AU-C (43 kDa). Phorbol myristate acetate, known to increase lymphokine
mRNA stability(10) , inhibited the induction of AU-B, providing
a correlation between the binding of this protein and increased mRNA
turnover(8) .
Apoptotic cells have an increased rate of RNA turnover, and it has been suggested that RNA degradation could play a role in the progression to cell death(11, 12) . In the immune system, apoptosis is thought to be involved in the elimination of potentially destructive, self-reactive immature lymphocytes (14) and excess effector lymphocytes that are generated in primary immune responses(13, 14, 15) . Apoptosis can be induced experimentally in T lymphocytes by withdrawal of growth factors, glucocorticoid treatment, heat shock, and UV-induced DNA damage or, in dividing cells, as a consequence of antigen receptor-mediated activation(13, 16) . An early marker of the death program is the activation of an endonuclease that cleaves chromosomal DNA between nucleosomes, yielding DNA multimers of 200 base pairs. DNA fragmentation is then followed by a variety of morphological changes that include condensation of the cytoplasm, swelling of the endoplasmic reticulum, plasma membrane blebbing, nuclear shrinkage, and the formation of dense chromatin masses known as apoptotic bodies. This process eventually leads to cell lysis.
Apoptosis can be induced in vitro in actively proliferating murine T cell hybridomas by
exposure to antigen or mAb directed against the TCR or the CD3
components of the TCR(17) . In this system, TCR signaling
initially results in a G/S cell cycle block followed by
genomic DNA fragmentation(18) . At low concentrations, drugs
that inhibit transcription and translation prevent the TCR-induced DNA
fragmentation, suggesting that the expression of new gene products is
required for TCR-induced apoptosis (19) . However, at higher
concentrations, inhibitors of transcription and translation cause DNA
fragmentation directly (18) through an unknown mechanism.
Here we show that in T cell hybridomas, TCR-generated signals result in the cytosolic accumulation of several previously described AU-specific RNA-binding proteins and a novel 70-kDa protein that is capable of binding the AU-rich element within the IL-2 3`-UTR as well a second site within the IL-2 coding region. The coordinate appearance of these RNA-binding proteins correlated with a shorter half-life of IL-2 mRNA, an increased rate of RNA degradation, and the induction of apoptotic cell death. It is therefore possible that RNA-binding proteins are involved in apoptosis-related RNA degradation.
Figure 1: Schematic representation of the RNA probes. Transcripts spanning different regions of IL-2 mRNA were obtained by SP6 polymerase-mediated in vitro run-off transcription from recombinant plasmids (described under ``Experimental Procedures'') linearized by the enzymes indicated in the figure. Whenever the IL-2 plasmid was linearized with SpeI or SspI, RNAs containing 4 or 2 AUUUA sequences were transcribed. RNA probes with no AUUUA sequences were obtained when the recombinant plasmids were linearized with TaqI, DdeI, or RsaI. The RNA IL-2 oligonucleotide was chemically synthesized and is restricted to the 3`-UTR ARE (indicated as the shadowed box). The c-myc cDNA was obtained by subcloning a double-stranded DNA oligonucleotide corresponding to the sequence that was previously shown to bind AU-A(8) . The sequence of the c-myc RNA probe obtained by in vitro transcription is shown. The results of UV cross-linking experiments performed with the different riboprobes and cytosolic extracts of activated T cells are summarized in the right panel of the figure.
Figure 2:
TCR-mediated signals induce a transient
expression of IL-2 mRNA in the A.AE7.2 T cell hybridoma. A,
the cells were stimulated with immobilized anti-CD3 mAb (CD3) for
the indicated times after which 20 µg of total RNA from each group
was analyzed by Northern blot with a labeled IL-2 cDNA. The specific
bands were detected by autoradiography. B, the half-life of
IL-2 mRNA was measured after either 3-4 or 6-8 h of TCR
stimulation. The cells were stimulated with immobilized anti-CD3 mAb
for the indicated time. Thereafter, new IL-2 mRNA transcription was
blocked by adding cyclosporin A, and the rate of IL-2 mRNA degradation
was measured as described under ``Experimental Procedures.''
The results represent the mean ± S.D. of four independent
experiments.
Cytosolic extracts from unstimulated T cells reproducibly contained proteins with molecular masses of 90, 65, 40-50, and 34 kDa that could be cross-linked to this RNA (Fig. 3A). Digestion of the reaction mixtures with proteinase K eliminated the labeled complexes, confirming that these factors are at least partially composed of protein (data not shown). Cytosolic extracts from T cells stimulated for 6, 8, or 10 h with anti-CD3 mAb reproducibly showed no change in the 65-kDa protein, a decrease in the 90-kDa protein, and an increase in the 34- and 40-50-kDa proteins when compared with extracts from resting T cells. The extracts from stimulated T cells also contained novel proteins of 70 and 30 kDa. An identical pattern was obtained with another independently derived T cell hybridoma (Fig. 3B). Although other bands were detected in some experiments, only the 90-, 70-, 40-50-, 34-, and 30-kDa proteins were reproducibly present in cytosolic extracts in all experiments, although the 30-kDa protein was not clearly resolved from the 34-kDa protein in some cases. Nuclear extracts from resting or activated T cells showed a complex pattern of RNA-binding proteins that only partially overlapped with the proteins detected in cytosolic extracts (compare Fig. 3C with 3A and 3B). The most prominent nuclear RNA-binding protein co-migrated with the 34-kDa protein observed in cytosolic extracts (Fig. 3C). Since the 34-, 30-, and 40-50-kDa proteins share many of the features of the AU-A, AU-B, and AU-C proteins described by Bohjanen et al. (8, see ``Discussion''), they will hereafter be referred to as AU-A, AU-B, and AU-C, respectively.
Figure 3:
Identification of RNA-binding proteins
able to bind RNA from the 3`-UTR of IL-2. A.AE7.2 (A, C) and DO.11.10 (B) cells were incubated in medium
alone (med) or with immobilized anti-CD3 mAb (CD3) for 2, 4, 6, 8, 10, and 24 h (A) or for 6
h (B and C); thereafter cytosolic (A and B) or nuclear (C) extracts were recovered. The
extract-equivalent of 10
cells was incubated with 2 fmol of
[
P]UTP-labeled transcript derived from the
3`-end of IL-2 (IL-2 SpeI). RNA-protein complexes were then
covalently linked by UV radiation, digested with RNase A, separated on
a 10% SDS-polyacrylamide gel, and detected by autoradiography. Arrows indicate the TCR-dependent RNA-protein
complexes.
As shown in Fig. 3A, the inducible RNA-binding proteins (70, 40-50, 34, and 30 kDa) were first detected after 6 h of TCR simulation and achieved maximal levels at 8 h. The amounts of the 40-50-, 34-, and 30-kDa proteins returned to basal levels after 24 h, whereas the 70-kDa protein remained detectable at this time point. Therefore, the appearance of the inducible RNA-binding factors correlated with the times at which IL-2 mRNA became less stable (compare Fig. 2B and Fig. 3A).
The appearance of the 70-kDa
complex following anti-CD3 mAb stimulation often correlated with a
decrease in the 90-kDa complex (Fig. 3A). This
suggested that the protein present in the 70-kDa complex could be
derived from the 90-kDa complex. Partial V8 protease digestion of the
70- and 90-kDa RNA-protein complexes (produced by UV cross-linking
using a P-labeled IL-2 RNA and cytosolic extracts from
resting or activated T cells) generated overlapping sets of peptide
fragments (Fig. 4). Thus, the proteins in the two complexes are
highly related to each other at least in their RNA binding domains.
Similar V8 protease experiments were performed on gel slices
corresponding to AU-A and AU-B. As shown in Fig. 4, these two
proteins showed distinct protease patterns as reported previously (9) and do not appear to be related to the 70- and 90-kDa
proteins.
Figure 4:
V8 protease mapping of the TCR-induced
RNA-binding proteins. Cytosolic extracts (10 µg) from unstimulated
cells and cells stimulated for 6 h with anti-CD3 mAb, were incubated
with a [P]UTP-labeled transcript from the IL-2
sequence (IL-2 SpeI; Fig. 1; 2
10
cpm) and processed as described in Fig. 3. Gel slices
corresponding to AU-A, AU-B, the 90- and the 70-kDa complexes were
excised from the gel, rehydrated, and reloaded on a 15% polyacrylamide
SDS gel. Different concentrations (0-1 µg) of V8 protease
were then loaded in each lane, and the digestion was performed in the
gel at room temperature for 30 min. The cleavage products were then
detected by autoradiography.
Figure 5:
Specificity of the AU-binding factors.
Five µg of cytosolic extracts derived from unstimulated cells (med) or anti-CD3 (CD3) mAb-stimulated cells
were incubated with 1.5 fmol of [
P]UTP-labeled
IL-2 riboprobe (IL-2 SpeI-4 AUUUA) in the absence (0)
or in the presence of a 10-fold (10), 100-fold (100),
or 500-fold (500) molar excess of the following unlabeled
competitors. A, transcripts derived from the IL-2 sequence
containing four AUUUA (SpeI), two AUUUA (SspI), no
AUUUA (RsaI); B, an IL-2 RNA oligonucleotide
restricted to the ARE sequence (IL-2) and transcripts
derived from the 3`-UTR of the c-myc cDNA (myc).
Following UV cross-linking, the RNA-protein complexes were digested
with RNase A, separated on SDS-polyacrylamide gel electrophoresis and
detected by autoradiography.
The 90- and
70-kDa complexes had a different specificity. The cross-linking of
these proteins to the P-labeled 278-base IL-2 SpeI riboprobe (see Fig. 1) that contains four AUUUA
elements was inhibited by the unlabeled IL-2 SpeI RNA (Fig. 5A) and by the 39-base RNA oligonucleotide that
also contains the 4 AUUUA elements (Fig. 5B). Although
these proteins could be cross-linked directly to the
P-labeled 39-base IL-2 RNA oligonucleotide (data not
shown), suggesting that they can bind to the minimal ARE sequence, they
were not competed as efficiently as AU-A and AU-B by the cold
oligonucleotide (Fig. 5B). In addition, unlabeled RNAs
containing two (IL-2 SspI) or no (IL-2 RsaI) AUUUA
elements inhibited the cross-linking of the 70- and 90-kDa proteins to
the
P-labeled IL-2 SpeI RNA (Fig. 5A), indicating that these proteins also bind to
a sequence outside the ARE. The 70- and 90-kDa proteins were not
degenerate RNA-binding proteins, however, in that their binding to the
P-labeled IL-2 SpeI RNA was not inhibited by an
unlabeled c-myc RNA that was an efficient inhibitor of the
binding of both AU-A and AU-B (Fig. 5B). These results
suggest that AU-A and AU-B proteins bind only to the ARE but do not
discriminate between IL-2 and c-myc mRNAs, whereas the 90- and
70-kDa bind specifically to IL-2 mRNA but appear to bind the ARE and
another site.
[P]UTP-labeled riboprobes
corresponding to various portions of the 3`-end of the IL-2 mRNA were
used to identify the non-ARE p90/p70 binding site. As shown in Fig. 6, and as summarized in Fig. 1, the 90- and 70-kDa
proteins, but not AU-A, -B, or -C, could be cross-linked to a
56-nucleotide segment spanning nucleotides 508-564 at the 3`-end
of the IL-2 coding region. In contrast, AU-A, -B, and -C, but not p90
and p70, bound to a c-myc RNA. All of the RNA-binding proteins
bound to an IL-2 probe spanning nucleotides 508-660 that contains
the ARE, and p90 and p70 could be cross-linked to this RNA better than
to the RNA spanning nucleotides 508-564 that lacks the ARE.
Together, our results show that p90 and p70 bind independently to two
different elements in the IL-2 mRNA, one encompassing nucleotides
508-564, and the other encompassing nucleotides 589-627
that contains the ARE. In contrast, AU-A, -B, and -C bind only to the
IL-2 ARE containing greater than 2 AUUUA repeats.
Figure 6:
The 90- and 70-kDa proteins bind to a
second site in the IL-2 coding region. A, RNA probes
corresponding to the HindIII-TaqI (TaqI),
the TaqI-DdeI (DdeI), the DdeI-RsaI (RsaI), and the DdeI-SpeI (SpeI) sequences of IL-2 coding
region were transcribed in the presence of
[P]UTP as indicated under ``Experimental
Procedures.'' B, 5 fmol of each RNA was analyzed by UV
cross-linking experiments as described in Fig. 3for the ability
to form specific complex with RNA binding proteins that were detected
by autoradiography.
Figure 7:
The appearance of the RNA-binding proteins
does not require new RNA and protein synthesis and correlates with
apoptotic cell death. Cells were treated with medium alone (med); anti-CD3 mAb in the absence (CD3) or
presence of 0.2 µg/ml of cyclosporin A (
CD3+csa); or 10 µg/ml cycloheximide (
CD3+cx) for 6 h (A). In panel B,
the cells were treated with medium alone (med); immobilized
anti-CD3 mAb (
CD3); cyclosporin A 0.2 µg/ml (csa); 10 µg/ml of cycloheximide (cx); or 5
µg/ml of actinomycin D (actD) for 6 h. In panel
C, the cells were left untreated (med) or treated with
0.5% azide (azide) or with anti-CD4 mAb and rabbit complement (CK) for 30 min at 37 °C. The inducible RNA-binding
proteins were detected as described in Fig. 3. Three fmol of
P-labeled IL-2 RNA probe were used in the binding
reactions. The molecular masses of the standards are expressed in kDa.
In panel D, genomic DNA was isolated from cells (5
10
) treated for 6 h with medium alone (med);
immobilized anti-CD3 mAb in the absence (aCD3) or in the
presence of 0.2 µg/ml cyclosporin A (
CD3+csa) or
10 µg/ml cycloheximide (
CD3+cx); 10 µg/ml
cycloheximide (cx) alone, or 5 µg/ml actinomycin D (actD) alone. The cells were lysed in 30 µl of 50 mM Tris-HCl (pH 8), 10 mM EDTA, 0.5% laurylsarcosinate (Life
Technologies, Inc.), 0.5 mg/ml proteinase K and incubated for 10 h at
50 °C. The mixtures were then digested with RNase A and loaded on a
1.8% agarose gel containing 0.5 µg/ml of ethidium
bromide.
Figure 8:
Dexamethasone induces DNA fragmentation
and the RNA-binding proteins in T cell hybridomas and in nontransformed
T cells. Hybridoma cells were stimulated with medium alone (med); immobilized anti-CD3 mAb in the absence (CD3) or in the presence of 0.2 µg/ml cyclosporin A (
CD3+csa) or 40 nM staurosporin (
CD3+stauro) or 1 µM dexamethasone (
CD3+dex) for 6 h; or 1 µM dexamethasone alone for 6 (dex 6 h) or 24 h (dex 24
h) (A, B). A.E7 T cells were stimulated with
medium alone (med) or with 1 µM dexamethasone (dex) for 24 h in the absence(-) or in the presence
(+) of 10 units/ml recombinant IL-2 (C, D). In panels A and C, the cells were lysed and genomic DNA
was analyzed in an ethidium bromide-agarose gel as described in Fig. 7. In panels B and D, the cytosolic
extracts were recovered, and the RNA-binding proteins were analyzed as
described in Fig. 3. Three (B) or 5 (D) fmol
of
P-labeled IL-2 RNA probe were used in the binding
reactions. The molecular masses of the
C-labeled standards
of panel B correspond to 200, 97.4, 68, 43, 29 kDa,
respectively.
Previous work by others demonstrated that TCR stimulation results in activation-induced cell death of T cell hybridomas(17) . Apoptosis depends on transcription and translation of new gene products as shown by the finding that at low doses, inhibitors of these processes prevent TCR-induced DNA fragmentation(19) . However, at high concentrations, the inhibitors themselves were shown to induce apoptotic cell death in T cell blasts(19, 28, 29) . Based on these results and those shown in Fig. 7, A and B, it was possible that the RNA-binding proteins were induced under any condition where apoptosis was induced, e.g. TCR signaling or exposure to high doses of protein synthesis inhibitors. To test this, we cultured a T cell hybridoma with anti-CD3 mAb, cycloheximide, or actinomycin D at doses that induced the cytosolic RNA-binding proteins and then analyzed genomic DNA for apoptosis-associated, oligonucleosomal cleavage. As shown in Fig. 7D, and as previously reported, TCR signaling resulted in DNA fragmentation that was inhibited by cyclosporin A ( (30) and Fig. 7D). Cycloheximide and actinomycin D, at 10 and 5 µg/ml, respectively, induced DNA fragmentation by themselves, suggesting that apoptosis was induced under these conditions. Therefore, cyclosporin A is capable of blocking the TCR-induced DNA fragmentation and the appearance of the cytosolic RNA-binding proteins, whereas TCR-mediated signaling alone and inhibitors of transcription and translation induce both genomic DNA fragmentation and the appearance of the RNA-binding proteins. Moreover, the accumulation of the most inducible RNA-binding factors, AU-B and p70, appeared to be specific for cells that were dying by apoptosis because they were not detected in the cytosol of cells undergoing necrotic cell death following antibody plus complement or azide treatment (Fig. 7C).
To further test the idea that the appearance of RNA-binding proteins correlated with apoptosis induction, we reproduced the previous finding (31) that glucocorticoids and TCR signaling each induce apoptosis in T cell hybridomas but that the combination does not (Fig. 8A). The inducible cytosolic proteins that could be cross-linked to IL-2 RNA (AU-B and p70) were detected only in cells undergoing apoptosis (cells treated with anti-CD3 mAb or dexamethasone, but not both) (Fig. 8B). Unlike TCR-mediated signals, dexamethasone treatment does not induce IL-2 mRNA expression (data not shown), thus disassociating the appearance of the RNA-binding proteins and lymphokine production. These results suggest that the inducible RNA-binding proteins probably bind to AU-rich RNAs in addition to IL-2 RNA.
We also tested whether the up-regulation of the RNA-binding
proteins correlated with apoptosis in nontransformed T cells. Treatment
of A.E7, a CD4 nontransformed T cell clone, with
dexamethasone resulted in both DNA fragmentation (Fig. 8C) and the appearance of the cytosolic
RNA-binding proteins (Fig. 8D). Moreover, addition of
the T cell growth factor IL-2 to the T cell cultures containing
dexamethasone inhibited both apoptosis induction (Fig. 8C) and the accumulation of the cytosolic
RNA-binding proteins (Fig. 8D).
Taken together, our results show that the cytosolic appearance of the RNA-binding factors and the development of an apoptotic phenotype were correlated in two different T cell types in response to a variety of stimuli.
Figure 9:
RNA degradation is accelerated in
apoptotic cells. A, the cells were labeled with
[H]uridine for 2 h and then treated with medium
alone (med); anti-CD3 mAb in the absence (aCD3) or in
the presence of 5 or 0.5 µg/ml actinomycin D (aCD3+actD 5,
aCD3+actD 0.5); actinomycin D alone (actD 5, actD
0.5); or with anti-CD3 mAb in the presence of 0.2 µg/ml
cyclosporin A (aCD3+csa). B, the cells
were labeled for 2 h with [
H]uridine (closed
circles) or with [
H]thymidine (open
squares) and then treated with the indicated concentration of
actinomycin D. After 8 h, cellular RNA (A, B) and DNA (B) contents were measured as described under
``Experimental Procedures.'' The results represent the mean
of triplicate determinations (the S.D. values was generally less than
10% of the mean) and are expressed as percentage of the RNA/DNA content
of cells incubated in medium alone where the RNA and/or DNA degradation
was considered to be zero.
TCR engagement elicits IL-2 production and then apoptotic cell death in T cell hybridomas. IL-2 mRNA is expressed shortly after TCR-stimulation, and its kinetic is shaped by effects on the rate of transcription of the IL-2 gene and by effects on stability of the IL-2 mRNA. Engagement of the TCR by anti-CD3 mAb quickly and transiently induces the expression of IL-2 mRNA that peaks around 4 h and rapidly disappears thereafter (Fig. 2A). Our results show that the IL-2 mRNA molecules present during the decline phase (i.e. after 4 h) are less stable than those present during the accumulation phase (i.e. before 4 h) (Fig. 2B), suggesting that the decline in IL-2 mRNA after its peak accumulation may be explained by cessation of transcription, as previously suggested(32) , and by decreased stability of the remaining mRNA. The observation that AU-rich sequences have been shown to confer instability on otherwise stable mRNAs(2) , and the fact that all of the inducible RNA-binding proteins appear in the cytosol during the period when IL-2 mRNA becomes less stable (compare Fig. 2and 3A), suggests but does not prove that these proteins play a role in targeting mRNA to rapid degradation.
Several of the proteins we describe here in a murine system are similar to proteins previously characterized in human T cells. Bohjanen et al.(8, 9) described three human proteins that bound to the ARE of the granulocyte-macrophage colony-stimulating factor 3`-UTR that they called AU-A, AU-B, and AU-C. Based on UV cross-linking and RNase T1 digestion experiments, these investigators reported molecular masses of 43, 40, and 50-60 kDa for AU-A, AU-B, and AU-C, respectively. However, because RNase T1 (8, 9) cuts RNA infrequently and leaves a relatively large fragment of RNA associated with the proteins, they calculated that the true molecular masses of AU-A, AU-B, and AU-C were 34, 30, and 43 kDa, respectively. These molecular masses correspond well with 34-, 30-, and 40-50-kDa RNA-binding proteins that we observed in extracts from murine T cells. AU-A and the 34-kDa murine protein are both present predominantly in the nucleus, although some of the protein is also present in the cytoplasm. Following TCR signaling, in both cases, a fraction of the nuclear protein translocates to the cytoplasm. Both AU-A and the 34-kDa murine protein bind to the c-myc and IL-2 3`-UTRs, and in the latter case they do so by recognizing at least three AUUUA sequences. AU-B and the 30-kDa murine protein are both present only in the cytoplasm and only after TCR signaling and, like AU-A, both bind to the IL-2 3`-UTR by recognizing at least three AUUUA sequences (Fig. 5). AU-C and the 40-50-kDa set of murine proteins, are a heterogeneous group of proteins that are present in the cytoplasm constitutively but increase after TCR signaling (Fig. 3). The specificity of AU-C is less well understood, although our competition results indicate that it has a relatively low affinity for the IL-2 3`-UTR. The simplest interpretation of these results is that the 34-, 30-, and 40-50-kDa proteins described here are the murine homologues of AU-A, AU-B, and AU-C. It should be noted, however, that we did observe a difference in the specificity of the 30-kDa murine protein from the one reported for human AU-B. Human AU-B was shown to not bind to the c-myc 3`-UTR (8) whereas the 30-kDa murine protein does. This discrepancy could be due to a subtle difference in the RNA-binding sites of the human and the murine forms of the protein or to differences in the binding conditions used in the two studies.
We also identified two novel, related proteins of 90 and 70 kDa that could be cross-linked to IL-2 mRNA. The 90-kDa protein is present in the cytosol of unstimulated cells, whereas the 70-kDa protein is detectable only after TCR signaling (Fig. 3, A and B). The findings that the appearance of the 70-kDa protein correlates with the disappearance of the 90-kDa protein (Fig. 3), and that the two proteins have overlapping V8 protease maps (Fig. 4) and RNA cross-linking specificities (Fig. 5) raise the possibility that the 70-kDa protein is derived from the 90-kDa protein. If this is the case, then the conversion of the 90-kDa protein to the 70-kDa protein must not require new RNA or protein synthesis because it occurs in the presence of inhibitors of these processes (Fig. 7B). It is possible that activation induces a post-translational conversion of the 90-kDa form to the 70-kDa from via phosphorylation, glycosylation, or proteolytic cleavage.
The simplest explanation for our UV
cross-linking results is that the 90- and 70-kDa proteins bind to the
AUUUA minimal element in the 3`-UTR and to a second site in the IL-2
coding region (Fig. 6). V8 protease mapping experiments indicate
that the domain on the 90-kDa protein that binds to the non-AUUUA
sequence is the same as the one that binds to the ARE. ()The
capacity of this protein to bind more than one site on the IL-2 mRNA
could allow it to form multimers, a property that could be important
for its function. Cooperation between two different instability regions
in the same mRNA has been previously described for the c-fos mRNA. As in the case of p90 and p70, one binding site is located
in the c-fos coding region, and the other in the ARE present
in the 3`-UTR. These elements appear to function by initiating the
deadenylation of the mRNA as an early step in c-fos mRNA
decay(33, 34) .
AU-A, AU-B, AU-C, and the 70-kDa protein were coordinately regulated. All appeared and disappeared at roughly the same times after the initiation of TCR signaling (Fig. 3) and all were dependent on the activity of calcineurin and protein kinase C (Fig. 8B). In addition, the induction of all four proteins by TCR signaling could occur in the presence of RNA or protein synthesis inhibitors (Fig. 7). This observation suggests that these proteins are already present in resting T cells but are unable to bind RNA until a TCR-dependent signal confers on them the binding activity. The idea that TCR-mediated signals could be involved in controlling RNA processing is supported by a recent finding that demonstrates a physical association between the signal transduction molecules p95vav (35) and c-Src (36) and the hnRNP-K.
Conversion to the RNA-binding forms of these proteins in the absence of new protein synthesis could be achieved by post-translational modification of the proteins, translocation of the proteins from a storage compartment to the cytoplasm, already described for the heterogeneous nuclear ribonucleoprotein A(37) , or by turnover of a labile inhibitor of the binding activity. Post-translational modifications of RNA-binding proteins have been described for the hnRNP A, B, and C proteins. The two modifications described for these proteins are phosphorylation of serine and threonine residues and methylation of arginine residues (for review, see (38) ). Phosphorylation and a redox switch have also been suggested for the activation of the 36-kDa protein expressed in Jurkat cells(39) . Of particular relevance to the current study is the finding that the 70-kDa protein component of the U1 small nuclear ribonuclear protein is proteolytically cleaved in apoptotic cells(40) . Moreover, post-translational changes in subcellular compartmentalization have already been proposed for the regulation of endonuclease activity during apoptosis induction(41, 42) . DNase I, a calcium, magnesium-dependent endonuclease of thymocytes and lymphocytes that is capable of cleaving DNA to oligonucleosome-size fragments, is thought to be bound in an inactive state to G-actin in the rough endoplasmic reticulum(42, 43) . It has been proposed that calcium-dependent changes in the rough endoplasmic reticulum cause DNase I to dissociate from G-actin and enter the nucleus where it could cause DNA fragmentation. It is thus possible that a labile inhibitor sequesters or inhibits the RNA-binding proteins until apoptosis is induced.
Dexamethasone also induced the appearance of the cytosolic
RNA-binding proteins (Fig. 8). This result raises the
interesting possibility that the induction of specific RNA-binding
proteins by dexamethasone is responsible for the
glucocorticoid-mediated destabilization of target mRNAs described in
lymphocytes (10) and fibroblasts(44) . The observation
that the increased turnover of interferon- in
dexamethasone-treated cells is mediated by the AU-rich sequence found
in the 3`-UTR (44) is consistent with this idea.
All of the
agents that caused the cytosolic appearance of the RNA-binding proteins
also induced apoptosis ( Fig. 7and Fig. 8). This
correlation held for stimuli as diverse as glucocorticoids, anti-CD3
mAb, and high dose RNA or protein synthesis inhibitors, that are each
likely to induce apoptosis via different signaling pathways. Recently
it has been reported that autocrine stimulation through the Fas
receptor is essential for TCR-activation induced T cell death (45, 46, 47) and that Fas probably signals
via the ceramide pathway. Interestingly, cyclosporin A inhibits the
induced expression of Fas ligand(45) , and glucocorticoids
inhibit the expression of Fas(47) , perhaps explaining how
these agents could block TCR-induced apoptosis. In contrast, oxidants,
DNA-damage, and glucocorticoids probably trigger apoptosis by different
signaling pathways. However, these diverse signals are thought to
converge on a common death pathway that involves the activation of a
protease called interleukin-1 converting enzyme, which is
antagonized by the Bcl-2 family of survival proteins (for review, see (48) ). If the RNA-binding proteins described here are involved
in apoptosis, then they must be activated at a point after the diverse
cytotoxic signals have converged. The observation that apoptosis
induced by TCR activation, like other routes to cell death, depends on
the activity of a serine or cysteine protease (49) suggests
that the induction of interleukin-1
converting enzyme or proteases
like it is one such point. It is conceivable that activation of
interleukin-1
converting enzyme-like proteases is responsible for
the conversion of p90 to p70 that we observed in apoptotic T cells. As
reported above, this would not be unprecedented because it has been
reported that a specific proteolytic cleavage of the 70-kDa protein
component of the U1 small nuclear ribonuclear protein is induced in
apoptotic cells(40) . Once activated, the RNA-binding proteins
could facilitate the induction of apoptosis, perhaps by facilitating
the turnover of AU-rich mRNAs that encode proteins that antagonize
death, such as growth factors or oncogenic proteins. Alternatively, the
RNA-binding proteins could play a more general role in RNA degradation
to ensure that all new transcripts are eliminated in dying cells.