(Received for publication, July 12, 1995; and in revised form, September 27, 1995)
From the
Gene rearrangement during the ontogeny of T- and B-cells
generates an enormous repertoire of T-cell receptor (TCR) and
immunoglobulin (Ig) genes. Because of the error-prone nature of this
rearrangement process, two-thirds of rearranged TCR and Ig genes are
expected to be out-of-frame and thus contain premature terminations
codons (ptcs). We performed sequence analysis of reverse
transcriptase-polymerase chain reaction products from fetal and adult
thymus and found that newly transcribed TCR- pre-mRNAs
(intron-bearing) are frequently derived from ptc-bearing genes but such
transcripts rarely accumulate as mature (fully spliced) TCR-
transcripts. Transfection studies in the SL12.4 T-cell line showed that
the presence of a ptc in any of several TCR-
exons triggered a
decrease in mRNA levels. Ptc-bearing TCR-
transcripts were
selectively depressed in levels in a cell clone that contained both an
in-frame and an out-of-frame gene, thus demonstrating the allelic
specificity of this down-regulatory response. Protein synthesis
inhibitors with different mechanism of action (anisomysin,
cycloheximide, emetine, pactamycin, puromycin, and polio virus) all
reversed the down-regulatory response. Ptc-bearing transcripts were
induced within 0.5 h after cycloheximide treatment. The reversal by
protein synthesis inhibitors was not restricted to lymphoid cells, as
shown with TCR-
and
-globin constructs transfected in HeLa
cells. Collectively, the data suggest that the ptc-mediated mRNA decay
pathway requires an unstable protein, a ribosome, or a ribosome-like
entity. Protein synthesis inhibitors may be useful tools toward
elucidating the molecular mechanism of ptc-mediated mRNA decay, an
enigmatic response that can occur in the nuclear fraction of mammalian
cells.
T-cell receptor (TCR) ()and immunoglobulin (Ig) genes
undergo programmed rearrangement events during lymphocyte ontogeny.
During this process, variable (V) elements are juxtaposed to joining
(J) elements to create functional genes(1, 2) . In
some TCR and Ig genes, diversity (D) elements are also included in this
rearrangement process. The tremendous combinatorial possibilities
afforded by this rearrangement mechanism permit the generation of a
wide variety of antigen receptors. Additional variability is provided
by the enzyme terminal transferase which introduces random nucleotides
at the junctions between V, D, and, J elements(1, 2) .
Variability is also engendered by the low fidelity of the rearrangement
event itself; the borders of each element are not fixed, sometimes
leading to small deletions at the junctions between the V, D, and J
elements. The collective result of these insertional and deletional
events is that a large fraction of rearrangement events will generate
out-of-frame (nonproductive) genes that contain premature termination
codons (ptcs).
Since out-of-frame TCR and Ig genes are commonly generated during normal lymphocyte development, there may exist a mechanism that diminishes the expression of these nonfunctional ptc-containing genes. Consistent with this hypothesis, most Ig and TCR cDNA clones obtained from cDNA libraries have been in-frame(3, 4, 5, 6) . Studies with cultured cells have provided evidence that out-of-frame Ig transcripts are down-regulated compared with in-frame transcripts (7, 8, 9, 10) . In addition, several other genes exhibit depressed mRNA levels when mutated to contain ptcs ((11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) ; reviewed in (27) ). One approach toward analyzing mechanisms responsible for ptc-mediated decay is to study this event in simpler eukaryotic organisms that are more amenable to genetic analysis. Toward this end, it has been shown that Saccharomyces cerevisiae and Caenorhabditis elegans down-regulate ptc-bearing mRNAs (28, 29, 30, 31, 32) . In S. cerevisiae, the decay of ptc-bearing mRNAs appears to be a cytoplasmic event. Several genes have been identified (the upf and smg series) that participate in ptc-mediated mRNA decay in S. cerevisiae and C. elegans(28, 29, 30, 31, 32) . Determination of the precise functional role of the upf and smg proteins may contribute greatly to our understanding of this novel regulatory process.
Vertebrates may use a ptc-mediated
decay mechanism that differs from the down-regulatory mechanism in
lower eukaryotic cells. In vertebrates it has been shown that many
ptc-bearing mRNAs are degraded in the nuclear fraction, rather than the
cytoplasmic fraction. Preferential nuclear degradation has been
demonstrated for ptc-bearing triosephosphate isomerase, dihydrofolate
reductase, -globin, v-src, major urinary protein (MUP),
and Ig transcripts (9, 12, 14-17, 20, 23, 24, 26). This is an
unexpected observation, since termination codons are generally
considered to be recognized only by ribosomes in the cytoplasm. Several
models have been put forward to explain how ptcs could affect nuclear
events (see ``Discussion''). In order to elucidate the novel
mechanism that is responsible for the recognition and decay of
ptc-bearing mRNAs in vertebrates, it is necessary to identify a system
that efficiently down-regulates such aberrant mRNAs. In addition, the
identification of an approach to reverse the down-regulatory response
would be a useful tool toward understanding the underlying mechanism
and its associated components.
In our previous work (33, 34) we showed that mature mRNA derived from a
transcriptionally active TCR- gene in the SL12.4 T-lymphoma cell
clone failed to accumulate in the cytoplasm, although TCR splicing
intermediates were easily detectable in the nucleus. We showed that
incubation of SL12.4 cells with several different protein synthesis
inhibitors dramatically induced the accumulation of mature TCR
transcripts in the cytoplasm(35) . In the present study, we
show that the rearranged TCR-
gene in the SL12.4 T cell clone is
out-of-frame and therefore contains ptcs. We then demonstrate in
transfection experiments that the inducibility by protein synthesis
inhibitors is exclusively restricted to ptc-bearing TCR-
transcripts. Protein synthesis inhibitors efficiently reversed the
down-regulatory effect of ptcs in any of a number of TCR exons. The
reversal of ptc-mediated mRNA decay by protein synthesis inhibitors was
a general response that was not restricted to particular stages of
T-cells, nor was it restricted to lymphoid cells. The ability to
reverse this response may be useful toward studying the molecular
mechanism of nonsense codon-mediated decay in the nucleus of vertebrate
cells.
Since nonfunctional ptc-bearing TCR genes are generated as a
part of normal thymic ontogeny, we then asked if this down-regulatory
mechanism operates in normal T-cells in vivo. We show that
although ptc-bearing TCR- genes are transcribed normally and give
rise to normal levels of pre-mRNA in the thymus, that the
levels of mature mRNA derived from these nonfunctional genes
is selectively depressed. This constitutes the first in vivo evidence in higher eukaryotes that the ptc-mediated mRNA decay
pathway selectively depresses mature mRNA levels, not pre-mRNA levels.
Our findings reveal new insights into the ptc-induced down-regulatory
mechanism and serve to further generalize the importance of this
mechanism.
HeLa cells were stably transfected with 5-20 µg of DNA by calcium phosphate precipitation(37) , and individual stably transfected clones were isolated for Northern blot analysis. Transient transfections were performed by electroporation with an Invitrogen Electroporator II according to the manufacturer's instructions (Invitrogen Corp., San Diego). Total cellular RNA was harvested 2 days after transfection. For polio virus infection, near confluent HeLa cells were infected with polio virus type I (Mahoney strain) at a concentration of 75 plaque-forming unit/cell. The virus was incubated with the HeLa cells for 30 min at room temperature, followed by addition of Dulbecco's modified Eagle's medium without serum (designated time point 0 of the infection). Total RNA from HeLa cells was prepared as described(33) . Cytoplasmic RNA was prepared by lysis in 0.6% Nonidet P-40, 0.15 M NaCl, 10 mM Tris (pH 8), 0.1 mM EDTA. RNA was electrophoresed, blotted, and probed as described(34) . The relative amount of RNA loaded per lane was assessed by methylene blue staining of the rRNA content (38) as well as hybridization with the housekeeping genes cyclophillin and CHO-A(39) .
To obtain the sequence of the VJ junctional region in
the VC
transcript in the SL12.4
cell clone, cDNA was prepared using 1 µg of RNA from SL12.4 cells
treated for 6 h with CHX (100 µg/ml). Oligo(dT) was used as a
primer for cDNA synthesis, and the oligonucleotides V
and E2 were used for PCR.
The proportion of out-of-frame pre-mRNA TCR-
transcripts that we found in the adult thymus differed strikingly from
the value we determined for TCR-
mature mRNA. Of 28
independent mature cDNAs sequenced, none were out-of-frame (Table 1). The samples sequenced included V
,
V
, and V
transcripts, as well as
random V
transcripts detected by anchored PCR.
The
proportion of in-frame and out-of-frame TCR transcripts was also
determined in day 16 fetal thymus. At day 16 of gestation, the thymus
is dominated by immature double-negative
(CD4CD8
) and double-positive
(CD4
CD8
) thymocytes that might be
subject to different regulation than more mature thymocytes. Our
analysis revealed that TCR-
pre-mRNAs were commonly out-of-frame
(38%), but mature mRNAs rarely were (4%; Table 1). Collectively,
the results show that both fetal and adult thymus actively transcribe
in-frame and out-of-frame TCR-
genes but selectively depresss the
levels of mature out-of-frame transcripts.
Figure 1:
Down-regulation of TCR-
transcripts that possess stop codons in internal exons. Upper
panel, the V
constructs transfected into SL12.4
cells: construct A, pIF; construct B, pFS1; construct C, pFS2; construct D, pFS3. Lower
panel, Northern blot analysis of cytoplasmic RNA (10 µg) from
stably transfected SL12.4 cells incubated in the presence or absence of
cycloheximide (CHX). Hybridization with the V
probe shows the expression of the transfected genes, while
hybridization with the cyclophillin probe shows that the blots are
equally loaded.
Since the basis for the
decrease in mRNA levels may be due to recognition of the premature
nonsense codon by a ribosome, we tested whether the addition of protein
synthesis inhibitors could reverse the down-regulation. Incubation with
the protein synthesis inhibitor CHX dramatically induced mRNAs derived
from the three constructs that possessed ptcs (Fig. 1). In
contrast, CHX had little or no effect on mRNA expression from the
in-frame construct (Fig. 1). Thus, incubation with CHX
specifically reversed the down-regulation of out-of-frame TCR-
transcripts.
Two of the out-of-frame VDJ constructs
shown in Fig. 1were generated by adding a 10-nucleotide XhoI linker at different sites within the exon. To test if the
reading frameshift caused by the XhoI linker was depressing
expression, we tested the effect of adding three XhoI linkers
to the normal gene (construct A); this would maintain the correct frame
by introducing 30 nucleotides. The construct containing three XhoI linkers at the EcoRV site of the VDJ
exon was expressed at high levels, regardless of the presence of
CHX (data not shown). In contrast, when four XhoI linkers were
introduced at the EcoRV site so that the construct was now
out-of-frame, the expression pattern was the same as the construct with
a single XhoI linker (e.g. CHX-inducible).
Next, a
nonsense codon was introduced into an in-frame TCR- gene to
determine if a ptc could trigger the down-regulatory response without a
frameshift. A UAA nonsense codon was introduced in place of a UAU codon
within the VDJ
exon. This single nucleotide mutation
was sufficient to efficiently down-regulate mRNA levels (Fig. 2). Treatment with CHX reversed the down-regulation (Fig. 2). As a control, the UAU codon was converted to a
synonomous UAC codon. This construct expressed high levels of TCR-
mRNA regardless of whether CHX was present or absent (Fig. 2).
Figure 2:
A premature nonsense codon is sufficient
to trigger down-regulation. Northern blot analysis of total cellular
RNA (2 µg) obtained from SL12.4 cells stably transfected with pUAA,
a V construct that possesses a ptc at amino acid
position 50, or pUAC, a construct that possesses a UAC codon at this
same position. Hybridization with the V
probe shows
the expression of the transfected genes, while hybridization with the
cyclophillin probe shows that the blots are equally
loaded.
To determine whether the down-regulatory mechanism displayed stage
specificity in its effects, an out-of-frame V gene
was transfected into two T-lymphoma cell clones (RS4.2 and AKR1) that
display a more mature phenotype than the SL12.4 cell clone used in our
study. Although the RS4.2 cell clone has a double negative phenotype
(CD4
8
) like SL12.4 cells, it
constitutively expresses TCR-
transcripts and displays a pattern
of cell surface markers indicative of a more mature phenotype (36, 45) . The AKR1 clone exhibits an even more mature
phenotype: it is a double-positive cell clone that constitutively
expresses both TCR-
and -
mRNA(33) . We observed that
the transfected out-of-frame V
gene was expressed at
very low levels in stably transfected lines of either RS4.2 or AKR1
cells (Fig. 3). CHX treatment induced the out-of-frame
V
transcript in both cell lines (Fig. 3).
Thus, the RS4.2 and AKR1 T-cell clones exhibit the same down-regulatory
response as the less mature SL12.4 cell clone.
Figure 3:
Down-regulation of TCR- transcripts
in T-cells at different stages of maturation. Northern blot analysis of
cytoplasmic RNA (10 µg) obtained from RS4.2
(CD4
CD8
) or AKR1
(CD4
CD8
) T-lymphoma cells stably
transfected with the out-of-frame V
construct
pFS1-1. Hybridization with the V
probe shows the
expression of the transfected genes, while hybridization with the
cyclophillin (cyclo) probe shows RNA loading. Note that the
lane from CHX-treated AKR1 cells is underloaded, as demonstrated with
the cyclophillin probe.
Figure 4:
Differential expression of in-frame and
out-of-frame TCR- transcripts in the SL12.4 T cell clone. Upper panel, the nucleotide sequence of the out-of-frame
TCR-
mRNA transcribed from the endogenous
V
C
gene in the SL12.4 cell clone. Lower left panel, the positions of the stop codons in the
endogenous out-of-frame V
C
gene and
the transfected in-frame V
C
gene. Lower right panel, Northern blot analysis of cytoplasmic RNA
(10 µg) prepared from SL12.4 cells stably transfected with the
in-frame V
C
construct
(pIF).
To further confirm allelic
specificity, TCR- mRNA expression was examined in the BW5147 cell
clone, which possesses a productively rearranged V
gene and a nonproductively rearranged V
gene(46) . Northern blot analysis showed that levels of
the mature 1.3-kb V
transcript were high, while the
1.3-kb V
transcript was barely detectable (at least
a 30-fold difference in expression; data not shown). This further
supports the notion that the down-regulatory mechanism discriminates
between in-frame and out-of-frame transcripts within a single cell.
Figure 5:
The
down-regulation of TCR transcripts bearing premature nonsense codons in
stably and transiently transfected HeLa cells. A: construct A, pAc/IF; construct B, pAc/FS2; construct C, pAc/IF. All TCR constructs are driven by a
human
-actin promoter (first exon shown) and all contain the first
-actin intron. B, Northern blot analysis of cytoplasmic
RNA (2 µg) from HeLa cells stably transfected with the constructs
shown. CHX-treated cells were incubated for 2 h with the drug.
Hybridization with the V
probe shows the expression
of the transfected genes, while hybridization with the CHO-A probe
shows the amount of RNA loaded. C, Northern blot analysis of
of cytoplasmic RNA (2 µg) from HeLa cells stably transfected with
construct B. D, Northern blot analysis of total cellular RNA
(10 µg) from HeLa cells transiently transfected with constructs A
(9 µg), B (9 µg), and/or C (3 µg) for 2 days. The cells
were incubated with CHX for 2 h unless otherwise
indicated.
We next assessed
whether transiently transfected TCR- genes are subject to the same
down-regulatory response as stably integrated TCR-
genes. To our
knowledge, a comparative study of the ptc-mediated regulatory mechanism
in transiently and stably transfected cells has not been reported
previously. HeLa cells were chosen for the transient transfection
experiments, since they are more efficiently transfected than SL12.4
cells. Fig. 5D (left panel) shows that an
out-of-frame TCR-
construct was expressed at dramatically reduced
levels compared with the in-frame construct in transiently transfected
HeLa cells. The out-of-frame construct was also expressed at low levels
relative to a co-transfected in-frame TCR-
gene that contained a
deletion (construct C, Fig. 5A) to permit independent
analysis of its expression (Fig. 5D, right panel). CHX
only weakly induced the out-of-frame transcripts after either a 2 h or
4 h (Fig. 5D) incubation in these transiently
transfected cells. We conclude that although the down-regulatory
mechanism is able to act efficiently on ptc-bearing transcripts derived
from ``free DNA'' in transiently transfected cells, CHX is
not able to efficiently reverse this down-regulatory response.
Figure 6:
Protein synthesis inhibitors with
different mechanisms of action all reverse the down-regulatory
response. Northern blot analysis of cytoplasmic RNA (2 µg) from
HeLa cells stably transfected with construct B from Fig. 5. The
cells were incubated with cycloheximide (100 µg/ml), pactamycin (3
µg/ml), anisomycin (100 µg/ml), emetine (300 µg/ml), and
puromycin (300 µg/ml), or medium alone for 2 h. Hybridization with
the V probe shows the expression of the transfected
genes, while hybridization with the CHO-A probe shows the amount of RNA
loaded.
Figure 7:
Polio virus infection selectively induces
ptc-bearing TCR transcripts. Northern blot analysis of cytoplasmic RNA
(2 µg) from HeLa cells stably transfected with constructs A and B
(from Fig. 5) followed by infection with polio virus.
Hybridization with the V probe shows the expression
of the transfected genes, while hybridization with the CHO-A probe
depicts the expression of a control
transcript.
Figure 8:
Cycloheximide reverses the down-regulation
of -globin transcripts that possess a premature nonsense codon. Upper panel: construct A, pGLOB (wild type
-globin construct); construct B, pGLOB39 (
-globin
construct that possesses a ptc at position 39). Lower panel,
Northern blot analysis of cytoplasmic RNA (2 µg) obtained from HeLa
cells stably transfected with pGLOB or pGLOB39. Hybridization with the
-globin probe (exon 2) shows the expression of the transfected
genes, while hybridization with the CHO-A probe shows that the blots
are equally loaded.
We have shown that ptcs trigger a diminution in mature
TCR- transcript levels in fetal and adult thymocytes in vivo (Table 1) and in transfected T-cells cultured in vitro (Fig. 2). Out-of-frame TCR genes that bear ptcs are
commonly generated by programmed rearrangement during lymphocyte
development. Most T-cells possess a nonproductively rearranged
TCR-
gene on one chromosome and a productively rearranged
TCR-
gene on the other chromosome(2) . Thus, it is
critical that the down-regulatory mechanism exhibit allelic
specificity: that it only depress the expression of the ptc-bearing
gene, not the functionally rearranged gene. Our demonstration that the
down-regulatory mechanism is indeed allelic-specific (Fig. 4)
suggests that this mechanism may be biologically relevant to the immune
system. Consistent with our results, Maquat's laboratory has
shown that the down-regulation of ptc-bearing triosephosphate isomerase
transcripts is allele-specific (11) and that ptcs act in
cis, not in trans, to down-regulate triosephosphate
isomerase mRNA levels (23) . A general down-regulatory
mechanism may be present in all cell types, where it serves to protect
cells from dominant negative mutations that result from mutant nonsense
codons. Since mutant proteins of the dominant negative class can be
potent inhibitors of the corresponding wild-type protein(49) ,
it is likely that there has been selection pressure to evolve a
mechanism to reduce the expression of such deleterious proteins. In the
case of the TCR-
protein, it is known that a truncated
amino-terminal version that has lost the ability to bind to CD3
molecules gains the ability to be secreted (50) and thus could
potentially interfere with immune reactions.
It is not clear how the
down-regulatory mechanism can distinguish a premature termination codon from a bona fide termination codon. One
possibility is that the termination codons which trigger
down-regulation lie upstream of an intron. This model is consistent
with the fact that most normal stop codons in mammalian genes
lie in the terminal exon (51) and hence would not be
followed by an intron. Evidence for this model is that ptcs in any of
the internal TCR- exons we tested, including the penultimate exon,
all result in strongly reduced expression (Fig. 1). Furthermore,
removal of the introns downstream of a ptc in the TCR-
gene
reversed the down-regulatory response. (
)Similarly, it has
been shown that removal of introns downstream of ptcs in the
triosephosphate isomerase gene inhibits the degradation of the encoded
message(14, 21) . If nonsense codon-mediated decay is
intron-dependent, then it is reasonable to suppose that the
down-regulatory mechanism involves the nucleus. In fact, several
reports have provided evidence that the presence of ptcs in transcripts
causes their decay in the nuclear fraction of mammalian cells, not in
the cytoplasmic fraction (9, 12, 14-17, 20, 23, 24, 26). We have
also found this to be the case for TCR-
transcripts, based on
nuclear subcellular fractionation studies and cytoplasmic half-life
measurements.
Two models have been proposed by Urlaub et al.(12) to explain how translation signals may affect nuclear mRNA stability. The translational translocation model proposes that translation of mRNA in the cytoplasm is first initiated while mRNA is still traversing through the nuclear pore. According to this model, if the ribosome encounters a ptc, export of the mRNA is interrupted, leading to degradation of the mRNA while it is still associated with the nucleus. In contrast, the nuclear-scanning model proposes that a codon scanner exists in the nucleus that searches for ptcs in mRNAs. Recognition of a ptc by the codon-scanner leads to nuclear degradation of the mRNA. More recently, a third model was suggested by Cheng & Maquat (19) in which recognition of a ptc by a cytoplasmic ribosome causes the transmission of a signal to the nucleus, triggering the degradation of the ptc-bearing mRNA in the nucleus. The key difference between these models is the locality of ptc recognition. For the translational translocation and the signaling models, recognition occurs in the cytoplasm, whereas the nuclear scanning model proposes that recognition of nonsense codons takes place in the nucleus.
Given that the down-regulation of many mRNAs occurs
in the nuclear fraction of mammalian cells, it is important to
determine if the regulation is exerted on pre-mRNA or mature mRNA. In
this study, we assessed this question in fetal and adult thymus and
determined that the presence of ptcs dramatically down-regulated mature
TCR- mRNAs, but had little or no effect on TCR-
pre-mRNA
levels (Table 1). This implies that the nuclear down-regulation
triggered by ptcs in vivo is not due to a decreased
rate of gene transcription or decreased stability of the primary
transcript. Thus, the most likely mechanisms by which ptcs depress
TCR-
mature mRNA levels are by: 1) inhibiting the splicing of one
or more introns present in TCR-
pre-mRNAs or 2) decreasing the
stability of partially or fully spliced TCR-
transcripts. Our
conclusions are consistent with several reports that show that ptcs
have no effect on gene transcription in cultured cell
lines(8, 12, 16, 19) . In contrast,
no consensus has emerged regarding the effect of ptcs on pre-mRNA
levels in cell lines; ptcs exert no discernible effect on
triosephosphate isomerase pre-mRNA levels and thus appear not to affect
splicing(19) , while ptcs apparently inhibit the splicing of
Ig-
chain and MVM pre-mRNAs(9, 15) .
Protein
synthesis inhibitors prevented the down-regulation of ptc-bearing TCR
transcripts in T-cells at different stages of maturation (Fig. 3) and in nonlymphoid cells (Fig. 5Fig. 6Fig. 7). In addition, we showed that
CHX treatment induced a reversal in ptc-mediated down-regulation of
-globin transcripts (Fig. 8). Consistent with our
observations, it was reported that CHX induces ptc-bearing iduronidase
transcripts in fibroblasts, as assessed by Northern blot
analysis(25) . In contrast, Lozano et al.(9) showed that CHX only marginally affects the levels of
ptc-bearing Ig-
transcripts, as judged by reverse
transcriptase-PCR analysis. The mechanistic basis for the apparent
difference between Ig and TCR regulation is not known. CHX may induce
events in B-cells that renders them still sensitive to the effects of
nonsense codons. Alternatively, B- and T-cells may utilize different
regulatory mechanisms to detect nonsense codons. Although this latter
hypothesis is formally possible, it seems unlikely, since we find that
protein synthesis inhibitors reverses the down-regulatory effect of
ptcs in HeLa cells (Fig. 5Fig. 6Fig. 7Fig. 8), as well as
T-cells(35) . In addition, the effect of CHX does not appear to
be context-specific, since CHX induced transcripts containing ptcs in
any of several different exons (Fig. 1).
We suggest that
protein synthesis inhibitors debilitate the down-regulatory mechanism
in one of two ways. One possibility is that protein synthesis
inhibitors directly prevent a ribosome in the cytoplasm or a
ribosome-like entity in the nucleus from reading mRNAs to determine if
they possess mutant nonsense codons. The rapid reversal of the
down-regulatory response by CHX treatment (Fig. 5C) is
consistent with this possibility. This hypothesis is also supported by
a study that showed that ptc-mediated mRNA decay is partially inhibited
by the presence of a stable hairpin loop in the 5` end of an mRNA or by
co-transfection with a nonsense suppressor tRNA(18) . Our
observation that protein synthesis inhibitors that act by different
mechanisms all efficiently reverse the down-regulation of
ptc-bearing TCR- transcripts (Fig. 6) suggests that either
a translocating ribosome or a modified translocating ribosome is
responsible. This entity is likely to contain components of the 60 S
ribosomal subunit, since anisomycin, CHX, and puromycin all affect the
function of this ribosomal subunit. Components of the 40 S subunit are
also likely to be involved, since emetine, which specifically binds the
40 S subunit, also reversed the down-regulatory response. Since many of
the inhibitors that we used are polysome stabilizers, it is possible
that they act by eliciting a build-up of ribosomes on ptc-bearing RNAs,
thus shielding these mRNAs from ribonuclease attack. This is unlikely
since the polysome destabilizers puromycin and pactamycin,
which generate naked ribosome-free transcripts, also induced
ptc-bearing TCR-
transcripts in HeLa cells (Fig. 6) and
T-cells(35) . Interestingly, puromycin is known to cause
premature termination of protein synthesis, yet like the other
metabolic inhibitors we used, it induced the accumulation of
ptc-bearing mRNAs. This suggests that the act of premature
translational termination is not sufficient to trigger the
down-regulatory response. Instead, recognition of a nonsense codon is
critical to engage the down-regulatory mechanism.
A second possible mechanism by which protein synthesis inhibitors reverse the down-regulatory response may be by preventing the translation of an unstable protein critical for the down-regulatory response. Since ptc-mediated down-regulation appears to involve the nucleus, an emerging possibility is that this response may not be mediated by a classical ribosome. An unstable protein(s) may be an essential component of this putative nonclassical ribosome. Alternatively, an unstable protein could be involved in putative ptc-mediated signaling events(19) , or it could mediate the degradation of ptc-bearing mRNAs. Our observation that ptc-bearing mRNAs are induced within 30 min after CHX treatment suggests that the half-life of such a labile protein would be relatively short. Evidence suggests that a labile protein also regulates the half-life of wild type c-fos transcripts(52) , although other evidence suggests that the rate of c-fos mRNA decay is regulated by other factors(53) . The nature of the labile proteins that may mediate the decay of unstable normal mRNAs (such as as c-fos) and aberrant mRNAs containing premature nonsense codons remains to be determined.
The use of various drugs to inhibit protein synthesis
allowed us to determine that de novo protein synthesis is
necessary for ptc-mediated degradation. However, their use does not
address the question of whether a cytoplasmic ribosome or a nuclear
ribosome-like entity is involved in this regulation, since these drugs
can accumulate in the nucleus as well as the cytoplasm. To address this
question, we chose to use the polio virus, since it replicates in the
cytoplasm of infected cells and selectively inhibits cytoplasmic
translation by cleaving a protein that is a component of one of the
initiation factors necessary for cap-dependent
translation(47) . Infection with polio virus resulted in an
increase in the levels of ptc-containing TCR- transcripts (Fig. 7). This result supports the notion that cytoplasmic ribosomes are involved in this mechanism. Hence, polio virus
infection could be preventing ribosomal recognition of ptcs in the
cytoplasm. Alternatively, polio virus may prevent the cytoplasmic
translation of an unstable protein destined for either the nucleus or
the cytoplasm that is necessary for ptc-mediated mRNA decay. One caveat
with interpreting this data is that since polio virus affects many host
processes(48) , it is possible that the reversal of the
down-regulatory response elicited by polio virus may be due to one of
its other activities.
Although CHX strongly induced ptc-bearing
TCR- mRNAs in stably transfected SL12.4 (Fig. 1Fig. 2Fig. 3Fig. 4) and HeLa cells (Fig. 5, A-C), CHX was a weak inducer in transiently
transfected HeLa cells (Fig. 5D). Thus, it appears that
ptc-bearing TCR-
transcripts are not efficiently rescued by
protein synthesis inhibitors in transiently transfected cells. Possible
explanations for this result include: (i) the declining rate of
transcription from DNA templates two days after transient transfection
may preclude significant increases in mRNA levels when protein
synthesis inhibitors are added at this time; (ii) the high level of
mRNAs transcribed from the large number of transiently transfected DNA
templates (in the small percentage of cells that efficiently
incorporate the exogenous DNA) may overwhelm the putative alternative
pathway for nuclear mRNA transport used when the ptc-scanning pathway
is blocked by protein synthesis inhibitors; (iii) the nonchromosomally
integrated DNA generated by transient transfection may be localized
inappropriately for the transcribed RNA to be efficiently exported from
the nucleus when the ptc-scanning pathway is blocked.
The level of down-regulation triggered by ptcs appears to vary depending on the gene. For example, TCR and Ig transcripts display a decrease in mRNA levels of 10-100-fold in response to ptcs ( (7, 8, 9, 10) and herein), while fully spliced triose-phosphate isomerase, v-src, and minute virus of mice transcripts are down-regulated by only 3-4-fold by the presence of ptcs(11, 14, 15, 24) . The basis for this difference is not known. Ptc-bearing TCR and Ig genes are generated as a part of normal lymphoid ontogeny, and thus it is possible that these genes have evolved cis-acting elements that improve their ability to be regulated by the ptc-mediated mRNA decay pathway. Since the rapid decay of some ptc-bearing mammalian transcripts appears to require introns(14, 15, 16, 17, 18, 19, 20, 21) , it is conceivable that splicing efficiency could influence the level of down-regulation induced by ptcs. TCR transcripts exhibit inefficient splicing (34) and thus may be localized to the nucleus for sufficient lengths of time to permit stringent scanning for ptcs.
Chromosomal context may be important for maximal down-regulation of
ptc-bearing transcripts. The out-of-frame
VJ
C
gene in
SL12.4 cells gave rise to almost undetectable levels of mature
transcripts; levels were induced by >50-fold after CHX treatment ( (33, 34, 35) and herein, Fig. 4). In
contrast, transfected out-of-frame TCR-
genes that presumably
integrated at nonhomologous sites gave rise to mature mRNAs that were
expressed at low-to-moderate levels and induced only 10-20-fold
by CHX (Fig. 1Fig. 2Fig. 3). Similarly, it has
been shown that endogenous out-of-frame Ig genes are expressed at
almost undetectable levels (up to 100-fold less than in-frame genes),
while transfected out-of-frame Ig genes are less dramatically
down-regulated(7, 8, 9, 10) . The
presence of ptcs depressed dihydrofolate reductase mRNA levels by
5-20-fold when the mRNA was derived from the endogenous gene, but
not at all when the mRNA was derived from stably transfected
dihydrofolate reductase genomic clones(12) . More recent
experiments suggest that the promoter dictates whether the
down-regulatory response is engaged: the
-globin and
cytomegalovirus immediate-early promoters permitted regulation, whereas
the HSV tk promoter did not(17) . Taken together, these results
suggest that nuclear context may strongly influence the down-regulatory
mechanism engaged by ptcs. The TCR-
gene is a useful model for
studying the molecular mechanism underlying this down-regulatory
network, since: (i) the TCR-
gene acquires mutant nonsense codons
during normal development; (ii) its expression is efficiently
suppressed by premature nonsense codons; and (iii) this regulation can
be manipulated by treatment with protein synthesis inhibitors.