By
From the Department of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report
our investigation of the mechanism for this downregulation using the TCR- gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-
exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full
coding sequence is not necessary for appropriate regulation. Mutation of the translation start
AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required
to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise
in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent
methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic
polypeptides from nonproductively rearranged genes.
Premature termination codons (PTCs)1 typically promote
a reduction in mRNA levels (for reviews see references
1, 2). Because termination codon recognition requires cytoplasmic ribosomes, it was anticipated that PTCs would
cause mRNA decay in the cytoplasm. One could envisage,
for example, that premature termination of translation in
the cytoplasm would leave the 3 The ability of nonsense codons to regulate nuclear RNA
metabolism is a paradox, because the only known entity
that can scan codons is a cytoplasmic ribosome. The TCR and
Ig genes are attractive gene systems for studying this enigmatic nuclear response to PTCs. Before their functional
expression, TCR and Ig genes undergo programmed DNA
rearrangements that permit them to encode a diverse number of receptors (17, 18). Part of this diversity is provided by the imprecise joining of the V, D, and J segments; further diversity is engendered by random nucleotide additions at the junctions of these gene segments by terminal
transferase. The consequence of these processes is that approximately two-thirds of the rearranged gene segments are
not in the proper translational reading frame, which results
in the generation of PTCs. It has been shown that nonproductively rearranged (PTC-bearing) Ig heavy and light
chain genes are underexpressed compared with their productively rearranged counterparts (19). The downregulation of Ig transcripts has been reported to occur posttranscriptionally (20) in the nucleus of B cells in vivo (22) and
B cell nuclear extracts in vitro (23).
We recently found that TCR- During the course of our studies we observed that the
downregulation of TCR- Plasmid Construction.
Construct A is identical to pAc/IF Cell Culture and Transfection.
HeLa cells were grown in
DMEM containing 10% fetal bovine serum. For transfection, the
cells (3 × 106 per 10-cm plate) were washed twice with Hepesbuffered saline (HBS; 140 mM NaCl, 0.75 mM Na2HPO4, 25 mM
Hepes, pH 7.05), resuspended in 250 µl DMEM without serum,
incubated at room temperature with 8 µg plasmid DNA (unless
other amounts are specified in figure legends) for 10 min, electroporated at 250 V using an Electroporator II (Invitrogen, Inc.),
and then incubated with 1 ml DMEM with 10% fetal bovine serum for 10 min in the cuvette before seeding on a 10-cm plate.
For transient transfections, RNA was isolated after 2 d in culture.
All transient transfections were repeated at least twice. For stable
transfections, the cells electroporated with a given plasmid DNA
sample were seeded onto three plates to generate three independent cell lines. Starting 1 d after transfection, stably transfected cells
were selected by incubating with the antibiotic G418 (800 µg/ml).
RNA Analysis.
Total cellular RNA was isolated as described
(26). Nuclear and cytoplasmic RNA was isolated by two different
methods. Method 1 relies on incubation in citric acid to lyse cells
and release intact nuclei (George Barker, Ph.D. thesis, 1992; details kindly provided by K. Beeman, The Johns Hopkins University,
Baltimore, MD). In this method, trypsinized cells from a 10-cm
plate were washed twice in HBS, resuspended in 1 ml 25 mM
citric acid, and then placed in a dounce homogenizer. After homogenization, cell lysis was judged by microscopy, typically, complete cell lysis required 5-10 strokes of a tight-fitting pestle. The
released nuclei were pelleted by centrifugation at 3,000 rpm for 2 min in a refrigerated microcentrifuge. The supernatant (cytosol) was combined with two or more volumes of denaturing guanidinium
isothiocynate buffer; cytoplasmic RNA was then prepared from
this cytoplasmic lysate by ultracentrifugation over a cesium chloride cushion, as described for total cellular RNA (26). The nuclear pellet was resuspended in 1 ml 0.25 M sucrose/25 mM citric
acid, dounced three times with a dounce homogenizer, underlaid
below 2 ml 0.88 M sucrose, 25 mM citric acid, and centrifuged at
3,000 rpm for 6 min. The supernatant was discarded, the nuclear
pellet was resuspended in 1 ml 0.88 M sucrose in RSB buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2), and the underlay step was repeated below 1 ml 0.88 M sucrose in RSB buffer.
After centrifugation, the supernatant was discarded and the pellet
was resuspended in guanidinium isothiocynate buffer, and nuclear
RNA was prepared from the nuclear lysate by ultracentrifugation
over a cesium chloride cushion, as described for total cellular
RNA (26). In method 2, the nuclear and cytoplasmic fractions
were obtained by cell lysis in the detergent NP-40, as described
(25). Cytoplasmic RNA was purified from the cytosol by the
means described for method 1. Nuclear RNA was prepared from
the nuclei after washing the nuclei with the stringent detergent sodium deoxycholate, as described (25).
portion of an mRNA
susceptible to nuclease attack. Consistent with this cytoplasmic decay hypothesis, PTCs have been shown to decrease the
half-lives of some mRNAs in the cytoplasmic compartment
of mammalian cells (3), and indirect evidence suggests
that the rapid decay of PTC-bearing transcripts in Saccharomyces cerevisiae occurs in the cytoplasm (6). Surprisingly,
many other reports have provided evidence that PTCs do
not appreciably affect cytoplasmic RNA half-life, but instead cause the degradation of mRNAs in the nuclear fraction of cells. Transcripts that have been reported to display
nuclear downregulation in response to PTCs include those
encoding dihydrofolate reductase (DHFR), triose phosphate isomerase (TPI), v-src, the mouse major urinary protein, and
-globin (9). Nuclear run-on analysis showed
that the decrease in nuclear mRNA levels imposed by PTCs is
not due to a decrease in the rate of gene transcription (9,
13, 14), thus implicating a nuclear posttranscriptional mechanism. The notion that nonsense codons regulate nuclear
posttranscriptional events is further supported by the observations that PTCs depress the splicing of transcripts from
the minute virus of mice (MVM) (15) and increase the accumulation of alternatively spliced fibrillin mRNAs that have
skipped the offending PTC (16).
transcripts are also subject to nonsense-mediated downregulation. Sequence analysis of pre-mRNA and mature mRNA RT-PCR products
amplified from mouse thymus demonstrated that out-offrame TCR-
genes (bearing PTCs) are actively transcribed but that fully spliced transcripts derived from these
genes do not accumulate (24). To study the mechanism for
this downregulatory response, we performed transfection
studies in cultured cell lines (24, 25). These studies showed
that frameshift or nonsense mutations that generate PTCs
cause a dramatic decrease in nuclear TCR-
mRNA levels. Intron deletion and addition studies demonstrated that
a PTC must be followed by at least one functional (i.e., spliceable) intron to trigger this downregulatory response.
This is consistent with a nuclear-based mechanism.
transcripts by PTCs could be
reversed by incubation with any of several different protein
synthesis inhibitors that possessed different mechanisms of
action: anisomycin, cycloheximide, emetine, pactamycin,
puromycin, and poliovirus (24, 25). This observation implicated the involvement of a ribosome or an unstable protein in the nuclear downregulatory response to PTCs. In
the present investigation, we further explored the possibility that the downregulation of PTC-bearing TCR-
transcripts involves a ribosome-based process. We found that
inactivation of initiator AUGs or expression of suppressor
tRNAs caused an increase in the levels of PTC-bearing
mRNAs in the nucleus, providing evidence that a translation-like surveillance mechanism exits that acts in the nuclear fraction of mammalian cells.
,
prepared as described (24). Site-directed mutagenesis was used to
introduce a missense (UAC) or a nonsense (UAA) codon at amino
acid position 50 in the VDJ exon of construct A to generate constructs B and C, respectively, using the oligonucleotides (oligo)
previously described (24). Construct D was made by mutating the
start AUG in construct A to an AUC using the sense oligo
MDA-154 (5
-GTTCTGAGATCTGCTCCAGAC-3
with the
mutated portion underlined). Construct E was made by mutating
the AUG at nt position 6 of exon 2 in construct A to a GUG, using the sense oligo MDA-155 (5
-CAGAACACGTGGAGGCTG-3
with the underlined portion mutated). Construct F was
generated by replacing exon 2 (AUG+) in construct D with the
AUG
mutant form of exon 2 in construct E. Constructs G-I
were made in an identical manner to constructs D-F, respectively, except that the parental vector used for site-directed mutagenesis was construct C. Construct J contained a UAG nonsense codon at amino acid position 50 in the VDJ exon as a result
of mutagenesis of construct A directed by the antisense oligo
MDA 247 (5
-GTCAGCGACATATGACTAATGGATC-3
with
the underlined portion mutated). The three Su+ tRNA constructs (pUCtSam, pUCtSoc, and pUCtSop) contain the tRNA genes cloned into the BamHI site of a pUC-based plasmid.
mRNA levels were assessed by hybridization with a 0.3-kb DNA
fragment corresponding to TCR-
exon 2 from the mini-gene (a
V
8.1 fragment kindly provided by Ed Palmer, Basel Institute for
Immunology, Basel, Switzerland). For stably transfected cells, the
relative amount of RNA loaded per lane was assessed by hybridization with the housekeeping gene CHO-A (28). For transiently
transfected cells, the neomycin gene probe was used to normalize
for transfection efficiency. The relative levels of TCR-
transcripts was calculated by values obtained from phosphoimage analysis of TCR-
and neomycin mRNA amounts.
To simplify the study of nonsensemediated regulation, we chose to generate mini-gene TCR-
constructs that contained the 5
portion of the third exon
(C
2.1) fused to the 3
end of the sixth exon (C
2.4). These
constructs contained three TCR-
exons: a V
8.1 leader exon
(exon 1), a rearranged V
8.1D
2 J
2.3 gene segment (exon 2),
and the fused C
2.1/2.4 exons (exon 3) (Figs. 1-4). The minigene constructs are driven by the
-actin promoter which
permits expression in HeLa cells. HeLa cells were chosen as
recipient cells for transfection because they are more efficiently transfected by electroporation than are the T cell lines we have tested. We have demonstrated before that HeLa
cells, like T cells, efficiently downregulate mRNA transcribed from transfected full-length, PTC-bearing TCR-
genes (24, 25).
To determine whether the mini-genes exhibited the
same downregulatory response to nonsense codons that we
had observed for the full-length gene, total cellular RNA
was prepared from HeLa cells transiently transfected with
three versions of the mini-gene differing only at codon position 50 in exon 2. Construct A had the normal UAU sequence at this position and thus possessed an open reading
frame extending to the stop codon in the final exon (see
schematic diagram in Fig. 4 A). Construct B contained a
UAC missense codon at this position and thus also had a
complete open reading frame. In contrast, construct C possessed a UAA nonsense codon at codon position 50 which
truncated the open reading frame (see Fig. 4 A). Northern
blot analysis of mature TCR- mRNA showed that constructs A and B were expressed at an equivalent level in
transfected HeLa cells that was >15-fold higher than that
of construct C (data not shown). Therefore, the TCR-
mini-gene system exhibited appropriate downregulation in
response to the UAA PTC.
Because evidence suggests that PTCs regulate
RNA metabolism in the nuclear compartment (9, 22,
23, 25, 32), the involvement of ribosomes has not been
clear. We used our mini-gene system to ask whether nonsense-mediated regulation requires a translation-like process. To determine whether an initiator AUG is required to
define a PTC, we performed AUG mutagenesis studies. In
addition to the initiator AUG in exon 1 that defines the
TCR- protein reading frame, we identified another inframe AUG in exon 2 that contained surrounding sequences (GAACACAUGG) similar to the Kozak consensus
sequence for efficient translation initiation (GCCGCCAUGG) (29). We mutationally inactivated either or both of these two AUGs and examined the effect on expression
from the in-frame mini-gene. Inactivation of the normal
start codon in exon 1 (construct D) caused a modest but reproducible two-fold decrease in mRNA levels relative to
the control (construct A) (Fig. 1). Similarly, inactivation of
the start AUG in exon 2 (construct E), or in both the first
and second exon AUGs (construct F) exhibited a two- to
threefold decrease in mRNA levels. We do not know the mechanism responsible for the modest reduction in expression from the in-frame AUG knockout constructs D-F.
Next we determined the effect of AUG inactivation on nonsense-mediated regulation. Mutation of the normal start codon in exon 1 (construct G) failed to significantly reverse the downregulatory effect of the PTC in this construct. Similarly, mutation of the putative start codon in exon 2 (construct H) had no measurable effect on mRNA expression. However, mutational inactivation of the AUGs in both exons 1 and 2 (construct I) strongly reversed the downregulatory effect of the PTC, permitting expression to increase to at least 50% of that of a corresponding nonPTC-bearing construct (construct F). This set of constructs (A, C-I) was also transiently transfected into baby hamster kidney (BHK) cells and the same results were obtained as in Hela cells (data not shown). We conclude that use of the start AUG in either exon 1 or exon 2 permits nonsensemediated regulation, but when both AUGs are rendered nonfunctional the nonsense-mediated downregulatory pathway is at best only partially engaged.
Because of the evidence that PTCs exert their effect in
the nuclear compartment of mammalian cells, we asked if
inactivation of start AUGs permitted reexpression of PTCbearing transcripts in the nucleus. Stably transfected cells
were used to prepare nuclear RNA because of the large
number of cells required. Fig. 2 shows data from highly purified nuclei isolated by the citric acid method (method 1 in
Materials and Methods); comparable results were obtained
using the NP-40/deoxycholate method (method 2; data not shown). We found that the AUG+ PTC-bearing gene
(construct C) accumulated a low amount of the 1.0-kb mature (fully spliced) transcript that was even lower in level
than the 2.8 kb pre-mRNA transcript. In contrast, the
double AUG knockout PTC-bearing gene (construct I)
expressed much higher steady state levels of mature mRNA
than pre-mRNA in the nuclear compartment. The ratio of
mature mRNA to pre-mRNA for construct I was comparable to that for the control non-PTC-bearing gene (construct F). Although the construct C-transfected cell line shown in Fig. 2 expressed somewhat higher levels of premRNA than the cell lines transfected with the other two
mini-gene constructs, we did not observe this difference
when other independent transfected cell lines were tested
(data not shown), nor have we observed that PTCs cause
measurable differences in the levels of pre-mRNAs transcribed from full-length TCR- genes (25). Cytoplasmic
RNA prepared in parallel with the nuclear RNA contained
only mature mRNA, and not pre-mRNA, as expected.
Cytoplasmic RNA showed the same relative expression
pattern for constructs C, F, and I as that of nuclear RNA.
The most straightforward interpretation of these results is
that inactivation of both AUGs affects neither transcription
nor pre-mRNA stability, but instead affects either premRNA splicing or mature mRNA stability in the nuclear
fraction of mammalian cells.
To determine whether the downregulation of TCR-
transcripts in response to PTCs involves codon recognition
by tRNA molecules, we co-transfected suppressor tRNA
constructs together with the TCR-
constructs shown in
Fig. 3 A. We tested a construct containing a UAA PTC
(construct C) by co-transfecting it with a plasmid encoding a suppressor tRNA specific for UAA (Su+ tRNA UAA).
The suppressor tRNA plasmid (12 µg) caused an eightfold increase in expression from construct C (4 µg) (Fig. 3 B).
As little as 4 µg Su+ tRNA UAA triggered a measurable
increase in expression from 4 µg construct C (data not shown).
We tested the specificity of the suppressor tRNAs on
mRNA levels. The UAA-specific suppressor tRNA that
upregulated expression from construct C had no significant
effect on expression from an in-frame gene (construct A)
(data not shown). To further investigate specificity, we analyzed the effect of UGA- and UAG-specific suppressor tRNAs on expression from the UAA-bearing gene (construct C). Little or no effect of these suppressor tRNAs on
mRNA levels was observed (Fig. 3 B). We also tested the
effects of suppressor tRNAs on a TCR- construct containing a UAG PTC (construct J). Co-transfection with the
UAG-specific suppressor tRNA plasmid upregulated expression from construct J by >10-fold (Fig. 3 C). In contrast, the UAA- and UGA-specific suppressor tRNAs failed
to increase expression from construct J.
We next determined if suppressor tRNAs act by regulating nuclear RNA metabolism. Highly purified nuclear
RNA was prepared from cells transfected with construct C
in the presence or absence of the Su+ tRNA UAA plasmid.
As shown in Fig. 4, the UAA-specific suppressor tRNA
caused an upregulation (fivefold) of mature mRNA (1.0-kb) levels in purified nuclei. In contrast, this suppressor tRNA
had no measurable effect on expression from a construct
lacking a PTC but otherwise identical to construct C (construct A). The suppressor tRNA also had no effect on the
levels of TCR- pre-mRNAs from either construct A or
C. Cytoplasmic RNA prepared in parallel with the nuclear
RNA displayed a pattern of mature mRNA expression similar to nuclear RNA. Collectively, the results suggest
that suppressor tRNAs induce an increase in the nuclear
levels of mature mRNA, and this, in turn, is reflected in
the cytoplasmic compartment.
A unique feature of T- and B-lymphocytes is the programmed rearrangements of the TCR and Ig genes necessary to elicit a selective immune response against foreign
antigens. A consequence of these rearrangement events is
the generation of a high proportion of out-of-frame TCR
and Ig genes that possess PTCs. Several reports have demonstrated that such PTC-bearing genes give rise to transcripts that are expressed at exceedingly low levels, as compared with their in-frame counterparts (19). In this
paper, we demonstrate that a TCR- mini-gene recapitulates this downregulatory response to PTCs. This TCR-
mini-gene provides a simple system to study nonsensemediated regulation. Our results with the mini-gene demonstrate that neither the full-length protein product of the
TCR-
gene nor the cis-acting elements in the interior of
the TCR-
gene are required for appropriate regulation.
More importantly, we were able to use this mini-gene system to ask whether nonsense-mediated downregulation
depends upon a translation-like process.
We found that mutational inactivation of the start AUG
and a downstream in-frame AUG in the TCR- minigene specifically reversed the downregulatory response to
PTCs in the nuclear fraction of mammalian cells (Figs. 1
and 2). To our knowledge, this is the first formal demonstration that inactivation of a start AUG triggers the increased accumulation of a transcript in the nucleus in vivo. Our results are consistent with those of Aoufouchi et al.
(23) who made similar observations using nuclear extracts
in an in vitro transcription-RNA splicing system. They
found that the inhibition of Ig
mRNA splicing by PTCs
is reversed by mutational inactivation of the start AUG
(23). Although we do not know whether TCR transcripts are also decreased in levels as a result of inhibited-splicing, regulation of TCR gene expression is clearly intron-dependent, based on our previous observation that at least one intron is required downstream of a nonsense codon to trigger
downregulation (25). In contrast, the v-src gene, which has
also been shown to depend on a start AUG to display decreased expression in response to PTCs in vivo (11), does
not contain introns and thus must engage the downregulatory response by an intron-independent mechanism.
Suppressor tRNAs reversed the downregulation of
TCR- transcripts in response to PTCs (Figs. 3 and 4),
thus strongly implicating a tRNA-dependent translationlike process in nonsense-mediated regulation. We tested
suppressor tRNAs specific for all three nonsense codons
(UAA, UAG, and UGA) and demonstrated that only suppressor tRNAs with the appropriate cognate anticodon sequence could reverse the downregulatory response. Our
results confirm and extend the findings of the Maquat laboratory, which showed that a UAG-specific suppressor tRNA
partially reversed the downregulation of TPI transcripts
bearing a UAG PTC (30). Because PTCs downregulated TPI transcript levels by only three- to fivefold, and since
the reversal mediated by the UAG suppressor tRNA was
only partial, Belgrader et al. (30) observed only a modest
twofold increase in transcript levels in response to the suppressor tRNA. We also found that suppressor tRNAs elicited
only a partial reversal of regulation but because TCR-
transcripts were so strongly downregulated by PTCs, we
observed a more dramatic induction of transcript levels by
suppressor tRNAs; in some cases, greater than 10-fold (Fig. 3).We demonstrated for the first time that suppressor tRNAs
exert their action on nonsense codon-bearing mRNA in
the nuclear compartment (Fig. 4). We also demonstrated
that this is a selective effect on fully spliced mRNA; premRNA levels were not measurably affected by suppressor
tRNAs, as assessed by quantitative northern blot analysis.
This latter result suggests that suppressor tRNAs affect neither gene transcription nor pre-mRNA stability. Because
we did not observe a decrease in cytoplasmic TCR-
levels in response to suppressor tRNAs, it is also unlikely that
suppressor tRNAs act by inhibiting nuclear-to-cytoplasmic
transport. Thus, the simplest explanation for our results is
that suppressor tRNAs increase the nuclear stability of
PTC-bearing, fully spliced transcripts. However, it is also
possible that suppressor tRNAs reverse an RNA splicing
block imposed by PTCs on TCR-
transcripts. Relief of
RNA splicing inhibition would normally be expected to
cause a measurable increase in pre-mRNA levels (a result
that we did not observe), but this would not occur if the
rate of nuclear RNA degradation was higher than the rate
of RNA splicing.
How is a translation-like mechanism able to downregulate transcripts in the nuclear fraction of mammalian cells?
One possibility is that cytoplasmic ribosomes adjacent to
the nuclear pore may direct the decay of PTC-bearing
mRNAs during their transit from the nucleus. Such a
translational-translocation or co-translational export model
(2, 9) is consistent with evidence that mRNAs exit the nuclear pore with 5 to 3
directionality (see reference 2 and
references therein). However, because RNA splicing appears to occur in the nucleoplasm proper, and not at the
nuclear pore (31), this model is not easily reconciled with
evidence that nonsense codons modulate RNA splicing
(15, 16, 22, 23). Further evidence against this model is the
finding that an intron must be downstream of a nonsense
codon to trigger the downregulation of TCR-
transcripts,
and that this intron will still engage the downregulatory response even when it is so close to the nonsense codon that
it must be spliced before the entry of the nonsense codon
into the cytoplasm (25). Still, while these lines of evidence argue against nuclear pore-associated ribosomes explaining
all effects of nonsense codons, it remains an attractive hypothesis that ribosomes spatially situated on the cytoplasmic
side of the nuclear envelope mediate some aspects of nonsense-mediated regulation.
A second possibility originally proposed by Urlaub et al. (9) is that a nuclear factor exists that scans and directs the decay of PTC-bearing mRNAs in the nucleus. This nuclear scanning model provides a simple explanation for why PTC-bearing mRNAs are degraded in the nuclear compartment. Nuclear recognition also explains why nonsensemediated regulation is intron-dependent (10, 25, 32) and how nonsense codons could regulate nuclear RNA splicing (15, 16, 22, 23).
What macromolecular factor could mediate nuclear surveillance? The evidence provided in this report, as well as those of others (11, 23, 30) supports the possibility that it is a ribosome. Also consistent with this notion is the observation that factors required for translation, including eIF-4E, accumulate in the nucleus (33). The site for nuclear ribosome scanning could be the nucleolus, where complete or nearly complete 40S and 60S ribosomal subunits reside (34). Some mRNAs are known to accumulate in nucleoli under some circumstances, consistent with the notion that the nucleolus is a site for mRNA trafficking (35). However, the low abundance of most poly(A)+ mRNA species in mammalian nucleoli (39), and lack of evidence that RNA splicing occurs in nucleoli, casts some doubt on the notion that nucleoli could be a site for nuclear surveillance of aberrant mRNAs.
Another possibility is that classical ribosomes do not mediate nuclear surveillance; instead a modified ribosome or
an entirely novel entity is involved. Recently, Aoufouchi
et al. reported that the ribosomal inhibitors puromycin and
cycloheximide failed to block the ability of B cell nuclear
extracts to discriminate between PTC-bearing and nonPTC-bearing Ig transcripts (23). Because neither puromycin nor cycloheximide act on the 40S ribosomal subunit,
their data suggested the involvement of either the 40S subunit or a novel factor. In contrast to their results, we found that protein synthesis inhibitors that possess different mechanisms of action, including cycloheximide, puromycin, anisomycin, pactamycin, emetine, and poliovirus, all reversed
the downregulation of PTC-bearing TCR-
transcripts in
intact cells (24, 25, 27, 40, 41). Because these inhibitors act
directly on ribosomal subunits or associated factors, one interpretation of our finding is that the recognition of PTCs
by a ribosome or a modified ribosome is blocked by incubation with the inhibitors. Another interpretation is that
addition of the inhibitors blocked the translation of one or
more unstable proteins that are part of a nuclear scanner or
that are involved in RNA decay.
Nonsense-mediated regulation could act as a surveillance mechanism to downregulate the expression of PTC-bearing mRNAs encoding truncated polypeptides that possess dominant negative properties. Because TCR and Ig genes commonly gain PTCs during normal lymphoid ontogeny, these may be important targets for this putative surveillance mechanism. The TCR and Ig variable region polypeptides which would be expressed in the absence of this surveillance mechanism, could interfere with the assembly or function of the full-length versions of these proteins expressed within the same cell. The notion of a nuclear-based surveillance mechanism that detects nonsense codons challenges our current understanding of gene expression. An important issue for future research will be to determine if this mechanism revolves around a classical ribosome, a modified ribosome, or a novel macromolecule that shares features with a ribosome.
Address correspondence to Miles F. Wilkinson, Department of Immunology, Box 180, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX, 77030.
Received for publication 22 October 1996 and in revised form 22 January 1997.
1Abbreviations used in this paper: BHK, baby hamster kidney cells; DHFR, dihydrofolate reductase; HBS, Hepes-buffered saline; MVM, minute virus of mice; nt, nucleotides; oligo, oligonucleotide; PTC, premature termination codon; TPI, triose phosphate isomerase.We are grateful to Dr. John Capone (McMaster University, Hamilton, Ontario, Canada) for the suppressor tRNA constructs, and to Phillip Morris for providing assistance with some of the projects. We thank Mark Carter, Gilbert Cote, and Thomas Cooper for offering helpful comments on the manuscript.
This work was supported by grant GM39586 and fellowship GM18128 from the National Institutes of Health.
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