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Stability and Translation of TCR ζ mRNA
Are Regulated by the
Adenosine-Uridine-Rich Elements in
Splice-Deleted 3 ′ Untranslated Region of ζ
-Chain
Bhabadeb Chowdhury, Sandeep Krishnan, Christos G.
Tsokos, James W. Robertson, Carolyn U. Fisher,
Madhusoodana P. Nambiar and George C. Tsokos
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 177:8248-8257; ;
doi: 10.4049/jimmunol.177.11.8248
http://www.jimmunol.org/content/177/11/8248
The Journal of Immunology
Stability and Translation of TCR ␨ mRNA Are Regulated
by the Adenosine-Uridine-Rich Elements in Splice-Deleted
3ⴕ Untranslated Region of ␨-Chain1,2
Bhabadeb Chowdhury,3 Sandeep Krishnan,3 Christos G. Tsokos, James W. Robertson,
Carolyn U. Fisher, Madhusoodana P. Nambiar, and George C. Tsokos4
T
he complex autoimmune disease systemic lupus erythematosus (SLE)5 is characterized by multiple disorders of
cellular and humoral immune responses (1, 2). T cells
from patients with SLE display an overexcitable phenotype that is
characterized by replacement of the TCR ␨-chain with the FcR␥
chain (3) and aggregation of lipid rafts on the cell surface membrane (4).
The TCR ␨ gene is located in chromosome 1q23.1 (5–7) an area
that has been assigned susceptibility for the development of SLE
(8 –10). It spans at least 31 kb, and the transcript is generated as a
spliced product of 8 exons that are separated by distances of 0.7 kb
to over 8 kb (11). Recently it has been described that TCR ␨
mRNA and protein expression is significantly reduced in SLE (12–
16). Nucleotide sequence analysis of the TCR ␨ mRNA showed
increased frequency of alternatively spliced forms missing various
exons in SLE T cells (13, 17). Analysis of the 3⬘ untranslated
region (UTR) showed a novel 344 bp alternatively spliced form
with a deletion of nucleotides from 672 to 1233 of exon VIII of the
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver
Spring, MD 20910, and Department of Medicine, Uniformed Services University of
the Health Sciences, Bethesda, MD 20814
Received for publication July 24, 2006. Accepted for publication August 31, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study is supported by the National Institutes of Health Grants R01 AI42269 and
R01 AR39501.
2
The opinions and assertions contained herein are private views of the authors and are
not to be construed as official or as reflecting the views of the Department of the Army
or the Department of Defense.
3
B.C. and S.K. contributed equally to this work.
4
Address correspondence and reprint requests to Dr. George C. Tsokos, Department
of Cellular Injury, Walter Reed Army Institute of Research, Building 503, Room
1A32, 503 Robert Grant Avenue, Silver Spring, MD 20910. E-mail address:
[email protected]
5
Abbreviations used in this paper: SLE, systemic lupus erythematosus; ARE, AUrich element; WT, wild type; UTR, untranslated region; CS, conserved sequence; m,
mutant (in mARE1, mARE2, and mCS).
Copyright © 2006 by The American Association of Immunologists, Inc.
TCR ␨-chain mRNA. The alternatively spliced form of TCR ␨
mRNA with 344 bp 3⬘ UTR was predominantly expressed in SLE
T cells compared with normal T cells (18). Several alternatively
spliced isoforms of the TCR ␨ mRNA with different nucleotide
sequences of the 3⬘ UTR also have been recently identified in
murine T cells (19).
The defective TCR ␨ protein expression in SLE T cells inversely
correlates with the level of TCR ␨ mRNA with alternatively
spliced 3⬘ UTR and directly with mRNA bearing the wild-type
(WT) 3⬘ UTR (20). This correlation indicates the existence of regulatory elements in the alternatively spliced region of TCR ␨
mRNA that are critical for its cellular expression. The regulation of
mRNA stability is often mediated by elements within the 3⬘ UTR
(21–23). In a recent report, we demonstrated that the destabilizing
effect of the alternatively spliced 3⬘ UTR that was identified as part
of the TCR ␨ mRNA is not gene-specific and may confer instability to other genes (24). This effect was not cell type specific,
suggesting that trans factors are not required and the destabilizing
effect is simply 3⬘ UTR length dependent.
The 3⬘ UTRs of eukaryotic mRNAs play an important role in
regulating gene expression at the posttranscriptional level by modulating nucleocytoplasmic mRNA transport, polyadenylation status, subcellular targeting, translation efficiency, stability and rates
of degradation (21, 25–28). The length of 3⬘ UTR observed in
human mRNAs may range from 21 nt to 8.5 kb with an average of
0.5– 0.7 kb (29, 30). The 3⬘ UTR of TCR ␨ mRNA is ⬃1 kb, which
is considerably longer than average, suggesting that it may have
one or more important roles in regulation of gene expression.
The molecular mechanisms that lead to destabilization of the
TCR ␨ mRNA with alternatively spliced 3⬘ UTR are currently
unknown. The 3⬘ UTR of mRNA contains cis-acting elements, for
example, adenosine-uridine (AU)-rich elements (AREs), that bind
to trans-activating factors and either stabilize or destabilize the
transcripts (22, 31). Sequence analysis of TCR ␨ mRNA indicates
the presence of ARE both in the deleted and the alternatively
spliced 3⬘ UTR. The TCR ␨-chain with splice-deleted 3⬘ UTR also
0022-1767/06/$02.00
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Systemic lupus erythematosus (SLE) T cells display reduced expression of TCR ␨ protein. Recently, we reported that in SLE
T cells, the residual TCR ␨ protein is predominantly derived from an alternatively spliced form that undergoes splice deletion
of 562 nt (from 672 to 1233 bases) within the 3ⴕ untranslated region (UTR) of TCR ␨ mRNA. The stability and translation
of the alternatively spliced form of TCR ␨ mRNA are low compared with that of the wild-type TCR ␨ mRNA. We report that
two adenosine-uridine-rich sequence elements (AREs), defined by the splice-deleted 3ⴕ UTR region, but not an ARE located
upstream are responsible for securing TCR ␨ mRNA stability and translation. The stabilizing effect of the splice-deleted
region-defined AREs extended to the luciferase mRNA and was not cell type-specific. The findings demonstrate distinct
sequences within the splice-deleted region 672 to 1233 of the 3ⴕ UTR, which regulate the transcription, mRNA stability, and
translation of TCR ␨ mRNA. The absence of these sequences represents a molecular mechanism that contributes to altered
TCR ␨-chain expression in lupus. The Journal of Immunology, 2006, 177: 8248 – 8257.
The Journal of Immunology
8249
contain a 31 nucleotide sequence (from 973 to 1003) that is conserved across the 3⬘ UTR TCR ␨ mRNA of several species (19)
implying a role in the stability of TCR ␨ mRNA. Species-conserved elements in the 3⬘ UTR of the AUF1 mRNA are important
in the regulation of AUF1 expression (32, 33).
Because the instability and the defective translation of the alternatively spliced 3⬘ UTR TCR ␨ mRNA contribute to the decreased expression of TCR ␨ protein in SLE T cells, we hypothesized that the 562-bp splice-deleted 3⬘ UTR of TCR ␨ mRNA
contains crucial cis-elements that bind factors that may confer stability and sufficient translation rate. When the region is spliced out
the mRNA become unstable and poorly translated. In this study,
we demonstrate that two AREs defined by the splice-deleted 3⬘
UTR are essential for the normal expression of TCR ␨-chain.
Therefore, the production of TCR ␨ mRNA with splice-deleted 3⬘
UTR represents a molecular mechanism that contributes to translational regulation of decrease expression of TCR ␨-chain in patients with SLE.
Materials and Methods
Materials, Abs, and cell culture
Unless indicated, all reagents used for biochemical methods were purchased from Sigma-Aldrich, Pierce, or Fisher Chemical. Enzymes for restriction digestion were obtained from New England Biolabs and Promega.
The TCR ␨ mAb 6B10.2, recognizing the amino acids 31– 45 of the
polypeptide (N-terminal mAb), was purchased from BD Pharmingen. The
C-terminal TCR ␨ mAb recognizing the amino acids from 145 to 161 is
described elsewhere (34). All cell culture reagents were obtained from
Invitrogen Life Technologies unless otherwise indicated. T cells were isolated from heparinized peripheral blood of six normal volunteers (three
men and three women, ages 18 – 40 years) by positive depletion of non-T
cells by magnetic separation (Miltenyi Biotec) as previously described
(35). The protocol has been approved by the Institutional Review Board.
PCR amplification and cloning of the splice-deleted 3⬘ UTR
TCR ␨ mRNA
Single-stranded cDNA was synthesized from total RNA by using the AMV
reverse transcriptase-based reverse transcription system from Promega and
oligo(dT) primer as instructed by the manufacturer. The primers were synthesized by Sigma-Genosys. The full-length TCR ␨ mRNA with WT 3⬘
UTR was amplified first by PCR using primers of 5⬘-AGC CTC TGC CTC
CCA GCC TCT TTC TGA G-3⬘ (sense bp 34 – 62 according to the numbering of Weissman et al. (6)) and 5⬘-CCC TAG TAC ATT GAC GGG
TTT TTC CTG-3⬘ (antisense bp 1472–1446). Then the full-length TCR ␨
mRNA with splice-deleted and alternatively spliced 3⬘ UTRs were amplified by PCR using specific primers designed for splice-deleted and alternatively spliced 3⬘ UTR. The sequences of splice-deleted primers are
5⬘-TAT TCC CCT TTA TGT ACA GGA TGC TTT GG-3⬘ (sense bp
672–700) and 5⬘-CCT GTA GCA CAT GGT ACA GTT CAA TGG TG-3⬘
(antisense bp 1205–1233). The various sequences of AU-rich regions of
106 bp of ARE1 (566 to 672), 300 bp of ARE2 (672 to 972), and 332 bp
of conserved sequence (CS) (901 to 1233) for 3⬘ UTR of TCR ␨-chain were
amplified by PCR with XbaI sites. The amplification was conducted using
a high fidelity PCR system from Boehringer Mannheim in a Biometra T-3
thermal cycler after initial denaturation at 94°C for 6 min, 33 cycles at
94°C for 1 min; 67°C for 1 min; 72°C for 2 min; and a final extension at
72°C for 7 min. The PCR products containing TCR ␨ with splice-deleted
(1135 bp) and alternatively spliced (916 bp) 3⬘ UTR were ligated to unidirectional pcDNA 3.1 His TOPO vector (Invitrogen Life Technologies).
Splice-deleted and alternatively spliced 3⬘ UTR TCR ␨-chain clones with
proper orientation were subjected to DNA sequencing from both orientations on an ABI 377 sequencer using ABI dye terminator cycle sequencing
kit (ABI PRISM; Applied Biosystems). WT clones were obtained from
normal T cells, whereas alternatively spliced clones were obtained from T
cells from patients with SLE as previously described (24).
Transfection of COS-7 cells with splice-deleted 3⬘ UTR TCR ␨
The COS-7 cells were subcultured in RPMI 1640 for 24 h before transfection containing 10% FBS and penicillin/streptomycin at 37°C in 5%
CO2 incubator. For transfection, cells were trypsinized, washed, and resuspended in 200 ␮l of Opti-MEM serum-free medium (Invitrogen Life
Technologies). Twelve micrograms of expression vector plasmid, pcDNA
3.1 V5 HIS TOPO containing TCR ␨-chain with splice-deleted or alternatively spliced 3⬘ UTR was added and electroporated at 250 V, 960 ␮F in
a 0.4-cm cuvette (Bio-Rad). Transfected cells were lysed at different time
points after incubation with actinomycin D (5 ␮g/ml) or cycloheximide (10
␮g/ml), and the mRNA was isolated after lysis of the cell membrane by
Nonidet P-40 (14).
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FIGURE 1. Molecular structure of the human TCR ␨ mRNA with splice-deleted as well as alternatively spliced 3⬘ UTR. A, The splice-deleted 3⬘ UTR
TCR ␨ mRNA generated by the splice deletion between 672 and 1233 bp at TCR ␨-chain. This splice-deleted TCR ␨ mRNA has a 562 bp but the WT and
alternatively spliced (AS) 3⬘ TCR ␨ mRNA have 906 bp and 344 bp at 3⬘ UTR. B, The nucleotide sequences of the primers used for the specific amplification
of TCR ␨-chain with splice-deleted and alternatively spliced 3⬘ UTR for semiquantitative RT-PCR. TCR ␨-chain with alternatively spliced 3⬘ UTR was
specifically amplified by a primer that spans both sides of the alternatively spliced site making it noncomplementary and will not anneal with the WT TCR
␨-chain. C, The nucleotide sequences of splice-deleted 3⬘ UTR TCR ␨-chain with 562 bp.
8250
REGULATION OF SPLICE-DELETED 3⬘ UTR TCR ␨-CHAIN
Real-time PCR of TCR ␨ mRNA
Site-directed mutagenesis of splice-deleted 3⬘ UTR TCR ␨-chain
Total RNA was prepared from transfected COS-7 cells and 1 ␮g of RNA
was reverse-transcribed into cDNA and diluted 10-fold for real-time quantitative PCR. The SYBR Green-based real-time quantitative PCR technique
was conducted with Cepheid Smart. The sequences of splice-deleted primers are 5⬘-TAT TCC CCT TTA TGT ACA GGA TGC TTT GG-3⬘ (sense
bp 672–700) and 5⬘-CCC AAG GCA GGG CCG TAA GCC CTG G-3⬘
(antisense bp 765–790). Alternatively splice primers are 5⬘-ACA GCC
AGG GGA TTT CAC CAC TCA AAG G-3⬘ (sense bp 566 –592) and
5⬘-CTT CAG TGG CTG AGA AGA GTG-3⬘ (antisense bp 650 – 671). The
condition for real-time PCR and calculation of the RNA concentration has
been previously described (24). Each sample was analyzed at two different
concentrations, and the result from the linear portion of the standard curve
was presented. Samples were analyzed in triplicate at each concentration,
and TCR ␨ mRNA with splice-deleted and alternatively spliced 3⬘ UTR
levels was normalized to the corresponding ␤-actin.
Site-directed mutagenesis was performed using the Quik-Change Site Directed Mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Three different mutants were used in the present study. Wildtype and splice-deleted 3⬘ UTR TCR ␨ point mutant (m) constructs were
generated by using the Quik-Change Site Directed Mutagenesis kit (Stratagene) of the TCR ␨ 3⬘ UTR expression vector construct. All three ATTTA
sites in the 3⬘ UTR of TCR ␨ cDNA were mutated to GGGTA (resulting
in 3⬘ UTR TCR ␨ mARE1, 3⬘ UTR TCR ␨ mARE2, and 3⬘ UTR TCR ␨
mCS). The three ATTTA to GGGT mutations were made at position 636
(resulting in 3⬘ UTR TCR ␨ mARE1), position 705 (resulting in 3⬘ UTR
TCR ␨ mARE2), and position 985 (resulting in 3⬘ UTR TCR ␨ mCS).
In vitro transcription and translation
cDNA encoding for the splice-deleted and alternatively spliced 3⬘ UTR
TCR ␨ was obtained by PCR amplication. The cDNA was used as the
template for the in vitro transcription of 3⬘ UTR TCR ␨. The splice-deleted
and alternatively spliced TCR ␨-chains were transcribed and translated using TNT T7 quick-coupled rabbit reticulocyte lysate transcription/translational system as recommended by the manufacturer (Promega). Plasmids (8
␮g) containing splice-deleted or alternatively spliced 3⬘ UTR were incubated with transcription/translation system in the presence of Transcend
biotin-lysyl-tRNA for 60 min at 30°C. The translated product was electrophoresed, transferred to polyvinylidene difluoride membranes, and the incorporated biotinylated lysine was detected nonradioactively by blotting
with streptavidin-HRP and developed using ECL chemiluminescent kit
(Amersham Biosciences).
Western blot analyses
Equal amounts (20 ␮g) of the total protein derived from cell lysate of each
sample were loaded in the gel and resolved by electrophoresis using
4 –12% bis-Tris NuPage gel (Invitrogen Life Technologies) under denaturing and reducing conditions and the proteins transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) (36). The immunoblot analysis was then conducted using specific Abs against TCR ␨
(clone 6B10.2). The binding was detected using an ECL system (Amersham Biosciences) according to the manufacturer’s instructions.
Luciferase reporter gene constructions and luciferase gene
expression assays
The full-length 3⬘ UTRs of splice-deleted, alternatively spliced, and the
various sequences of AU-rich regions (ARE1, ARE2, and CS) of 3⬘ UTR
TCR ␨-chain mRNAs were amplified by PCR with XbaI site and cloned
into the XbaI site downstream of the luciferase reporter gene in the pGL3Basic and enhancer vectors (Promega). The proper orientations of the
clones were verified by restriction mapping and sequence analysis. The
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FIGURE 2. Expression of TCR ␨ protein from
splice-deleted 3⬘ UTR following transfection into
COS-7 cells and following in vitro transcription and
translational analysis. The TCR ␨ with splice-deleted
and alternatively spliced (AS) 3⬘ UTR cloned into
pCDNA3.1/V5-HIS-TOPO expression vector and transfected to COS-7 cells by electroporation. The TCR ␨
with splice-deleted and alternatively spliced 3⬘ UTR
was transcribed and translated using TNT T7 quickcoupled rabbit reticulocyte lysate transcription/translational system from Promega. A, The specific probes
used in expression vector for transfection studies. B,
Western blot analysis of TCR ␨ protein(s) expressed in
transfected COS-7 cells by using TCR ␨-specific mAb
for experimental and ␤-actin Ab for control. C, Densitometric analysis of the immunoblots was done using
GEL-PRO software (Media Cybernetics), and the
amount of TCR ␨ protein produced in the transfected
cells has been shown in arbitrary units for the mean ⫾
SEM (n ⫽ 3). D, The translated product was lysed and
electrophoresed. Then it was transferred to polyvinylidene difluoride membranes and the incorporated biotinylated lysine in expressed protein was detected with
streptavidin-HRP and developed using an ECL chemiluminescent kit. E, Expression of TCR ␨ protein with
splice-deleted 3⬘ UTR. The blots were stripped and reprobed with TCR ␨-specific mAb. F, Densitometric
analysis of TCR ␨ protein produced for splice-deleted
and alternatively spliced 3⬘ UTR has been shown in
arbitrary units for the mean ⫾ SE (n ⫽ 3).
The Journal of Immunology
8251
luciferase constructs with various sequences of 3⬘ UTR TCR ␨ as well as
splice-deleted and alternatively spliced portions were transfected into
COS-7 cells or Jurkat cells and T cells in a 24-well plate (Corning) using
LipofectAMINE 2000 reagent (Invitrogen Life Technologies) (37) following the manufacturer’s protocol. Luciferase activity was determined using
a luciferase assay system (Promega) following the manufacturer’s protocol.
Briefly, the transfected cells were incubated in 6-well plates in a CO2
incubator at 37°C for 20 h and then cells were removed by scraping into
100 ␮l of reporter lysis buffer (Promega). Luciferase activity was assayed
with 20 ␮l of lysate and 80 ␮l of luciferase assay reagent (Promega) in a
TD20/20 luminometer (Turner Designs) using a commercially available kit
(Promega).
Densitometry and statistical analysis
Densitometric analysis of the Western blot was performed with the software program GEL-PRO (Media Cybernetics). Statistical analyses were
performed using the GraphPad Prism version 4.0 software and Minitab
version 13.
Results
We recently reported that SLE T cells express high amounts of an
alternatively spliced form of TCR ␨ mRNA that lacks 562 nt (672–
1233) in the 3⬘ UTR. The resultant TCR ␨ mRNA displays decreased stability and translation efficiency and thus contributes to
diminished expression of TCR ␨-chain in SLE T cells (20, 24)
implying a stabilizing role for the deleted region (Fig. 1).
To determine directly how the splice-deleted 3⬘ UTR affects the
expression of TCR ␨, we cloned the splice-deleted and alternatively spliced 3⬘ UTR TCR ␨ (Fig. 2A) into a eukaryotic expression
vector, pcDNA3.1/V5-HIS-TOPO, and then transfected the constructs into COS-7 cells and compared the expression of TCR ␨
protein in transfected cells by immunoblotting. As shown in Fig.
2B, TCR ␨ with splice-deleted 3⬘ UTR expressed a single band
with a molecular mass of 16 kDa. We observed that the level of
expression of TCR ␨ protein in COS-7 cells transfected with
splice-deleted 3⬘ UTR constructs was comparable to that observed
in cells transfected with WT but higher than that observed in cells
transfected with the alternatively spliced form. An anti-␤-actin Ab
was used to reblot stripped membranes and confirm equal protein
loading. The expression of TCR ␨ protein by alternatively spliced
3⬘ UTR was very low in comparison to splice-deleted form (Fig.
2B). Densitometric analysis of the immunoblots showed higher
amounts of TCR ␨ protein expression (⬎7-fold) in cells transfected
with splice-deleted 3⬘ UTR than alternatively spliced constructs
(Fig. 2C). These results suggest that splice-deleted 3⬘ UTR of TCR
␨ mRNA has a major impact on the expression of TCR ␨ protein.
In vitro translation of TCR ␨ with splice-deleted 3⬘ UTR
To rule out the possibility that higher production of TCR ␨ protein
in cells transfected with the splice-deleted constructs, we performed in vitro transcription and translation experiments with
splice-deleted and alternatively spliced 3⬘ UTR constructs using
biotinylated lysine as a nonradioactive label. In vitro transcription
and translation of TCR ␨-chain showed that TCR ␨ with alternatively spliced 3⬘ UTR construct produced significantly lower
amounts of protein than the splice-deleted 3⬘ UTR construct (Fig.
2D). We confirmed these results by stripping and reprobing the
blots by using TCR ␨-specific Ab (Fig. 2E). Densitometric analysis
showed that the level of expression of TCR ␨ protein in cells transfected with splice-deleted 3⬘ UTR resulted in a 10-fold increase
than the alternatively spliced 3⬘ UTR (Fig. 2F). These results
strongly support the presence of regulatory elements within the
splice-deleted 562-bp region of 3⬘ UTR TCR ␨ mRNA.
FIGURE 3. Stability of TCR ␨ mRNA in COS-7 cells transfected with
splice-deleted 3⬘ UTR by RT-PCR analysis. The TCR ␨-chain with splicedeleted and alternatively spliced (AS) 3⬘ UTR cloned and transfected to
COS-7 cells as described in Fig. 2. After 18 h of transfection, the cells were
incubated with transcription inhibitor actinomycin D (5 ␮g/ml) for 0, 2, 6,
and 10 h. Transfected cells were lysed and the total RNA was reverse
transcribed and PCR amplified using high-fidelity PCR system as described
in Materials and Methods. The splice-deleted and alternatively spliced 3⬘
UTR TCR ␨ mRNAs were quantitated by semi-quantitative RT-PCR.
Quantitation of the RT-PCR product was done by using GEL-PRO software, and the data are presented as a percentage of control. The data shown
are representative of three experiments with similar results from three different transfections. Error bars indicate SE.
Stability of TCR ␨ mRNA with splice-deleted 3⬘ UTR TCR ␨
To establish that the decreased stability of TCR ␨ mRNA with
alternatively spliced 3⬘ UTR was due to the lack of 562-bp splicedeleted 3⬘ UTR in SLE, we examined the stability of TCR ␨
mRNA that lacks this 562 bp in transfected COS-7 cells. Transfected cells were incubated with transcription inhibitor actinomycin D (5 ␮g/ml) for different periods of time (0, 2, 6, and 10 h) and
the levels of expression of TCR ␨ mRNA with splice-deleted and
alternatively spliced 3⬘ UTR were quantified by semiquantitative
RT-PCR analysis using specific primers as described in Materials
and Methods. There was no significant degradation of either
splice-deleted or alternatively spliced 3⬘ UTR TCR ␨ mRNA at 2 h
(Fig. 3). At 6 and 10 h, we recorded a mild reduction in the expression level of splice-deleted 3⬘ UTR mRNA, whereas at the
same time points, we observed a significant reduction of alternatively spliced form (6 h, p ⫽ 0.003; 10 h, p ⫽ 0.01) of TCR ␨
mRNA expression. These experiments demonstrate that splice-deleted 562-bp (nt 672–1233) segment of TCR ␨ mRNA contributes
to the stability of the TCR ␨ mRNA and that the stability of alternatively spliced form is due to the absence of these residues.
Next, we performed real-time RT-PCR to quantitate the stability
of TCR ␨ mRNA with splice-deleted and alternatively spliced 3⬘
UTR in transfected COS-7 cells treated with actinomycin D (5
␮g/ml) for 0, 2, 6, or 10 h to confirm the observed differences in
the stability of the TCR ␨ mRNA with splice-deleted and alternatively spliced 3⬘ UTR. The cDNA of reverse transcription product
of mRNA obtained from transfected cells at different time points,
was PCR amplified by using specific primers for splice-deleted and
alternatively spliced 3⬘ UTR TCR ␨ mRNA (Fig. 4) in Cepheid
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Expression of TCR ␨ protein by splice-deleted 3⬘ UTR TCR ␨
8252
REGULATION OF SPLICE-DELETED 3⬘ UTR TCR ␨-CHAIN
Thermocycler as described in Materials and Methods. The TCR ␨
mRNAs with splice-deleted and alternatively spliced 3⬘ UTR were
evaluated as the relative quantity against ␤-actin mRNA in the
cells before treatment with actinomycin D (data not shown). Realtime quantitative PCR confirmed our observations that splice-deleted 3⬘ UTR mRNA (splice-deleted 3⬘ UTR; 6 h, p ⫽ 0.05; 10 h,
p ⫽ 0.03) was more stable than alternatively spliced 3⬘ UTR
mRNA (alternatively spliced 3⬘ UTR; 6 h, p ⫽ 0.015; 10 h, p ⫽
0.021) and TCR ␨ with alternatively spliced 3⬘ UTR mRNA degraded to a greater extent compared with splice-deleted 3⬘ UTR in
transfected cells (Fig. 4). However, these results clearly demonstrate that the stability of TCR ␨ mRNA with splice-deleted 3⬘
UTR was higher and degraded to a lower extent than alternatively
spliced 3⬘ UTR in transfected COS-7 cell.
Restoration of translational efficiency of TCR ␨ mRNA by splicedeleted 3⬘ UTR
To determine whether the splice-deleted region that confers the
stability to TCR ␨ mRNA contributes also to its translation efficiency, we transfected to COS-7 cells with splice-deleted and alternatively spliced 3⬘ UTR of TCR ␨ constructs to examine the
levels of expression of TCR ␨ protein in transfected cells in the
presence of a protein synthesis inhibitor, cycloheximide (10 ␮g/
ml) for different periods of time (0, 2, 6, and 10 h). The levels of
expression of 16 kDa TCR ␨ protein by splice-deleted and alternatively spliced 3⬘ UTR were quantified by comparing with ␤-actin expression (Fig. 5). Although in transfected COS-7 cells, there
was no significant decrease in the TCR ␨ protein by 2 h for either
constructs, by 6 and 10 h we recorded no significant decrease in the
level of TCR ␨ protein expression with splice-deleted 3⬘ UTR in
comparison to alternatively spliced 3⬘ UTR (Fig. 5, A and B). The
level of expression of TCR ␨ protein with splice-deleted 3⬘ UTR
remained almost the same in transfected COS-7 cells even after
10 h of treatment with protein synthesis inhibitor, cycloheximide,
FIGURE 5. Restoration of translation efficiency of TCR ␨ protein by
splice-deleted 3⬘ UTR TCR ␨. The TCR ␨-chain with splice-deleted (SD)
and alternatively spliced (AS) 3⬘-UTR were cloned in expression vector
and transfected to COS-7 cells as described in Fig. 2. After 18 h of transfection, the cells were incubated with a protein synthesis inhibitor, cycloheximide (10 ␮g/ml) for 0, 2, 6, and 10 h. After treatment with protein
synthesis inhibitor, the transfected cells were lysed and immunoblots with
TCR ␨-specific Ab were prepared. A, Western blot analysis of TCR ␨ protein expressed with splice-deleted 3⬘ UTR TCR ␨ in transfected COS-7
cells by using TCR ␨-specific mAb for experimental and ␤-actin Ab for
control. B, Western blot analysis of TCR ␨ protein expressed with alternatively spliced 3⬘ UTR TCR ␨ in transfected COS-7 cells. C, Densitometric analysis of the immunoblots was done using GEL-PRO software
and the amount of TCR ␨ protein produced in the cycloheximide-treated
transfected cells has been shown in arbitrary units of the mean ⫾
SEM (n ⫽ 3).
whereas at the same time point, TCR ␨ protein expression with
alternatively spliced 3⬘ UTR was significantly decreased (Fig. 5C).
These experiments indicated that TCR ␨ with splice-deleted 3⬘
UTR restored the translational efficiency of TCR ␨ mRNA in transfected COS-7 cells.
Stability and expression of luciferase gene by splice-deleted 3⬘
UTR TCR ␨
We introduced the splice-deleted and alternatively spliced 3⬘
UTRs downstream of the luciferase gene to demonstrate whether
the splice-deleted 3⬘ UTR conferred stability to genes other than
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FIGURE 4. Stability and expression of TCR ␨ mRNA with splice-deleted 3⬘ UTR by real-time quantitative PCR analysis. Transfected COS-7
cells were incubated with transcription factor inhibitor actinomycin D (5
␮g/ml) as described for Fig. 3. The cells were lysed and the total RNA was
reverse transcribed, and the stability of TCR ␨ mRNA was determined by
real-time quantitative PCR. Real-time quantitative PCR was conducted in
Cepheid Smart Thermocycler using PCR beads by adding SYBR Green to
the reaction mixture. Real-time quantitative PCR analysis of TCR ␨ mRNA
stability in transfected cells with splice-deleted and alternatively spliced
(AS) 3⬘-UTR was conducted. A representative of three experiments with
similar results is shown. Error bars represent SE.
The Journal of Immunology
8253
Mapping of the stability regions within the splice-deleted 3⬘
UTR TCR ␨ mRNA
FIGURE 6. Luciferase activity of TCR ␨ with splice-deleted 3⬘ UTR.
The splice-deleted and alternatively spliced 3⬘ UTR of TCR ␨ were cloned
into XbaI site downstream of the luciferase gene in the sense orientation
and verified by restriction digestion. Luciferase construct containing splicedeleted 3⬘ UTR or alternatively spliced (AS) 3⬘ UTR was transfected into
COS-7 and Jurkat cells. A, Schematic representation of constitutive luciferase reporter constructs (not drawn to scale). Transcription (bent arrow)
was driven by the constitutive SV40 promoter upstream of the luciferase
reporter gene. B, PCR amplified products of splice-deleted or alternatively
There are three of these AREs (position 636, 705, and 985)
found in 3⬘ UTR TCR ␨. We introduced various sequences containing AREs of splice-deleted as well as alternatively spliced
3⬘ UTRs (106-bp ARE1, 300-bp ARE2, and 332-bp CS) (Fig.
7A) downstream of the luciferase gene to identify specific functional regions within the splice-deleted 3⬘ UTR that conferred
stability to genes other than that of TCR ␨-chain. Luciferase
constructs containing TCR ␨-chain with the indicated sequences
that contain ARE1, ARE2, and CS (which defines the third
ARE) were made by PCR amplification by engineering specific
primers containing XbaI site and cloned in pGL3-basic and enhancer vectors immediately after the luciferase gene at the 3⬘
region (Fig. 7B). The luciferase clones were confirmed by restriction mapping and sequence analysis (data not shown). The
resulting luciferase reporter constructs containing ARE1,
ARE2, and CS of 3⬘ UTR TCR ␨ were individually cotransfected with ␤-galactosidase to COS-7, Jurkat cells, and normal
T cells. As shown in Fig. 7C, the luciferase activities in transfected COS-7 cells were significantly decreased with the ARE1
of alternatively spliced 3⬘ UTR TCR ␨ construct compared with
ARE2 and CS of splice-deleted 3⬘ UTR TCR ␨ (COS-7; alternatively spliced to ARE2; p ⫽ 0.038 and alternatively spliced to
CS; p ⫽ 0.002). These results were reproduced when Jurkat
spliced 3⬘ UTR of TCR ␨-chain containing XbaI site. C, Verification of
luciferase clones by restriction digestion using XbaI. D, Luciferase activity
in transfected COS-7 cells with splice-deleted and alternatively spliced 3⬘
UTR. E, Luciferase activity in transfected Jurkat cells with splice-deleted
and alternatively spliced 3⬘ UTR. Data shown are the mean ⫾ SEM of the
three independent determinations (n ⫽ 3).
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that of TCR ␨-chain (Fig. 6A). Luciferase constructs containing
TCR ␨-chain with splice-deleted and alternatively spliced 3⬘
UTR were made by PCR amplification by engineered primers
containing XbaI site (Fig. 6B). PCR amplified products with
XbaI site were cloned into pGL3-basic and enhancer vectors
after digestion with XbaI, which is located immediately after
the luciferase gene at the 3⬘ region (Fig. 7A). The luciferase
clones were confirmed by restriction mapping that resulted in
appropriate size of inserts (562 bp for splice-deleted and 344 bp
for alternatively spliced form) (Fig. 6C). Finally, luciferase
clones were reconfirmed by sequence analysis (data not shown).
The resulting luciferase reporter constructs containing TCR ␨
splice-deleted or alternatively spliced 3⬘ UTR were individually
cotransfected with ␤-galactosidase to COS-7 cells. As shown in
Fig. 6D, the luciferase activity was significantly increased in
transfected COS-7 cells with the splice-deleted 3⬘ UTR construct compared with alternatively spliced 3⬘ UTR (COS-7
cells, p ⫽ 0.007). We further investigated the luciferase activity
in Jurkat and normal T cells transfected with splice-deleted and
alternatively spliced 3⬘ UTR. As shown in Fig. 6E, the luciferase activity was significantly decreased in Jurkat and T cells
transfected with alternatively spliced form compared with
splice-deleted 3⬘ UTR (Jurkat cells, p ⫽ 0.004). The empty
luciferase vector was used as a control. Maximal luciferase activity increase was found in transfected COS-7 (3.2-fold) cells
with spliced deleted 3⬘ UTR followed by Jurkat cells (2.8-fold)
and T cells (data not shown). These data indicate that splicedeleted 3⬘ UTR but not the alternatively spliced form confers
stability to other genes and mediates this effect independent of
the type of cell.
8254
FIGURE 8. Site-directed mutagenesis of AREs in 3⬘ UTR of TCR
␨-chain. Mutant constructs of 3⬘ UTR TCR ␨ were generated by using
the Quik-Change Site-Directed Mutagenesis kit (Stratagene). All three
ATTTA sites in the 3⬘ UTR of TCR ␨ cDNA were mutated to GGGTA
(resulting in 3⬘ UTR TCR ␨ mARE1, 3⬘ UTR TCR ␨ mARE2, and 3⬘
UTR TCR ␨ mCS). The three mutations were made at positions 636,
705, and 985, respectively (resulting in 3⬘ UTR TCR ␨ mARE1,
mARE2, and mCS). All three mutated and splice-deleted constructs
were transfected into COS-7 cells. After 20 h of transfection, TCR ␨
protein expression was measured by immunoblotting. A, Sequences of
3⬘ UTR TCR ␨-chain mRNA. AU-motifs are bold typeface. Special
regions representing WT nucleotides replaced with the mutant sequence
are individually named on the top. B, Western blot analysis of TCR ␨
protein expressed in transfected COS-7 cells. C, Densitometric analysis
of the immunoblots was done using GEL-PRO software and the amount
of TCR ␨-chain produced in the transfected cells has been shown in
arbitrary units of the mean ⫾ SEM (n ⫽ 3).
cells (Jurkat; alternatively spliced to ARE2, p ⫽ 0.04; alternatively spliced to CS, p ⫽ 0.001) were transfected with various
sequences of ARE region of 3⬘ UTR TCR ␨ (Fig. 7D). Maximal
increase was found in COS-7 cells (2.8-fold for ARE2 and
2-fold for CS) followed by Jurkat cells (2.9-fold for ARE2 and
1.8-fold for CS) and T cells (data not shown). These data
strongly support that ARE2 and CS in splice-deleted 3⬘ UTR
TCR ␨ mRNA that are absent in alternatively spliced form confer stability to TCR ␨ mRNA where as the ARE1 3⬘ UTR that
is only present in alternatively spliced form has no control in
regulating the TCR ␨ mRNA stability.
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FIGURE 7. The role of AU-rich regions in 3⬘ UTR TCR ␨ by reporter
gene expression. The various sequences of ARE1-, ARE2-, and CScontaining AREs of 3⬘ UTR TCR ␨ were cloned into XbaI site downstream of the luciferase gene in the sense orientation and verified by
restriction digestion as described for Fig. 6. A, Schematic representation
of different inserts (106 bp ARE1, 300 bp ARE2, and 332 bp CS) used
for luciferase reporter constructs. B, Schematic representation of constitutive luciferase reporter constructs (not drawn to scale). Transcription (bent arrow) was driven by the constitutive SV40 promoter upstream of the luciferase reporter gene. C, Luciferase activity in
transfected COS-7 cells with various sequences of AREs of 3⬘ UTR. D,
Luciferase activity in transfected Jurkat cells with various sequences of
AREs of 3⬘ UTR. Data shown are the mean ⫾ SEM of the three independent determinations (n ⫽ 3).
REGULATION OF SPLICE-DELETED 3⬘ UTR TCR ␨-CHAIN
The Journal of Immunology
Mutational analyses of splice-deleted 3⬘ UTR to identify the
specific gene sequences in controlling the translational
regulation of TCR ␨-chain
Discussion
In this study, we demonstrate that splice-deleted 562 bp within the
3⬘ UTR of TCR ␨ mRNA is directly involved in the positive regulation of transcription, stability, and translation of TCR ␨ mRNA.
Within this splice-deleted region, we have identified two novel
AREs that are responsible for mediating these effects. The alternatively spliced form of TCR ␨ mRNA that lacks these critical
elements is the predominant form of TCR ␨ mRNA observed in
SLE T cells. Our results demonstrate that the production of alternatively spliced forms of TCR ␨ lacking these critical residues in
its 3⬘ UTR region represents an important molecular mechanism
that contributes to reduced expression of TCR ␨-chain mRNA and
protein in SLE.
Regulation of mRNA stability is often mediated by elements
within the 3⬘ UTR (21–23). The 3⬘ UTR of mRNAs plays an
important role in regulating gene expression at the posttranscriptional level (21, 25–28). For example, a 171 bp region in the 3⬘
UTR of utrophin mRNA regulates its stability (38, 39). We examined the effect of the presence of the splice-deleted region on the
stability and translation of the luciferase reporter mRNA and established that the stabilizing effect of the splice-deleted 3⬘ UTR is
not gene-specific as it confers stability to other genes. Also, the
effect was found not to be cell type-specific, suggesting that either
trans factors are not required and the stabilizing effect is simply 3⬘
UTR length-dependent, or the required trans factors are non-cell
type-specific and universal in nature.
The role of ARE in mRNA stabilization and destabilization has
been studied intensively and are often found in the 3⬘ UTR of
short-lived mRNA of cytokines, transcription factors, and protooncogenes (40 – 42). Mutation of AUUUA motifs in the 3⬘ UTR of
IL-3 (43) and c-fos mRNA (44) results in increased stability of the
mRNA. The ARE elements in the 3⬘ UTR of ␤-catenin mRNA, a
well-known oncogene that plays a central role in the Wnt signaling
cascade, again contribute to its stabilization (45). AREs act as
mRNA instability determinants but also confer stabilization of the
mRNA by p38 pathway (46). ARE present in the 3⬘ UTR of various mRNA determine stability on instability by binding transactivating factors (22, 31).
The TCR ␨ with splice-deleted 3⬘ UTR contains a 31 nucleotide
sequence (from 973 to 1003) that is conserved across the TCR ␨
mRNA 3⬘ UTR of several species (19). We have provided evidence in this study that this conserved region is also involved in
the regulation of mRNA stability and expression because at position 985 it defines a third AUR. Therefore, the splice-deleted region defines two AREs (at positions 705 and 985) that are responsible for the stability and sufficient translation of the TCR ␨
mRNA. However, the ARE that is present upstream of the splicedeleted region (position at 636), has no role in the regulation of
TCR ␨ mRNA stability. This finding is interesting because AREs
may not be assigned functional roles in the absence of proper
documentation.
There are many reports describing the regulation of protein
expression mediated by the binding of proteins to the 3⬘ UTR
(47, 48), and splice deletion of TCR ␨-chain with alternatively
spliced 3⬘ UTR may abate the binding of these factors. Binding
of proteins to the myc-N and c-fos mRNA in human neuroblastoma cells results in increased mRNA stability and an aggressive clinical behavior of the tumor (49, 50). In contrast, association of polypyrimidine tract binding protein with the 3⬘ UTR
of the CD40L, a molecule that has been considered important in
the pathogenesis of human and murine SLE, promotes its instability (51–55). HuR, a nucleocytoplasmic shuttling protein,
has also been shown to bind mRNA-defined ARE and contributes to their stability (56, 57). Binding of proteins to mRNA
does not imply a functional effect. For example, although HuR
binds both IL-8 and GM-CSF mRNA-defined ARE, it stabilizes
only the GM-CSF mRNA (58) but not the IL-8 mRNA. How the
AREs within the 3⬘ UTR of TCR ␨-chain precisely regulate the
expression of TCR ␨ protein in T cells is currently under investigation in this laboratory.
In conclusion, we have identified two AREs within the 3⬘
UTR of TCR ␨ mRNA that are involved in the regulation of its
transcription, stability, and translation. Both of these AREs are
located within the splice-deleted region that is absent in TCR ␨
mRNA from patients with SLE. We have also shown that an
ARE located upstream of the splice-deleted region has no functional value. Therefore, specific molecular defects in SLE T
cells account for decreased TCR ␨ protein expression and abnormal T cell function.
Acknowledgments
We thank Dr. Anil B. Mukherjee, Section on Developmental Genetics,
Heritable Disorder Branch, National Institute of Child Health and Human
Development (Bethesda, MD) for helpful discussion and critical reading of
manuscript.
Disclosures
The authors have no financial conflict of interest.
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To provide further evidence that the ARE present in the splicedeleted 3⬘ UTR of TCR ␨ mRNA have a major impact on the
translation regulation of TCR ␨ mRNA expression that result in
higher production of TCR ␨ protein, we performed site-directed
mutagenesis. We have transcribed and translated TCR ␨ protein
with mutated constructs at different target region at AUUUA sites
(mARE1 at 636, mARE2 at 705, and mCS at 985 region) of 3⬘
UTR TCR ␨. A schematic representation of 3⬘ UTR TCR ␨ mRNA
including splice-deleted and alternatively spliced form and the position of the AUUUA motifs is shown in Fig. 8A. The sequences
of the respective 3⬘ UTRs are shown in Fig. 1C. There are three
AUUUA motifs in the 3⬘ UTR of TCR␨ mRNA. Motif 1 and motif
2 are located closely together at 636 and 705 bp, respectively, and
motif 3 at 985 bp. All these AUUUA motifs were mutated
GGGUA individually resulting in three mutated expression vectors. In this model, we have determined whether this mutation
would change the TCR ␨ protein expression in the transfected
cells. The TCR ␨ protein expression was studied by transfecting
mutant and splice-deleted WT 3⬘ UTR TCR ␨ vectors into COS-7
cells and measuring the expression of TCR ␨ protein levels by
Western blotting using TCR ␨ mAb (Fig. 8B). The splice-deleted
WT 3⬘ UTR vector was used as a control. Protein expression levels
of TCR ␨ were affected by the mutation at ARE2 and CS (ARE3)
but not at ARE1 of 3⬘ UTR TCR ␨ mRNA. As a control for protein
loading, these blots were stripped and reprobed with anti-␤-actin
Ab and expression of ␤-actin was noted to be normal in all experiments. Densitometric analysis (Fig. 8C) showed that the TCR
␨ protein expression in transfected cells with mutated mARE2 and
mCS was decreased ⬎3-fold than WT splice-deleted or mARE1,
indicating that the mutation at mARE2 or mCS in the 3⬘ UTR of
TCR ␨ mRNA results in induced down-regulation of TCR ␨ protein expression in transfected cells. These data also suggest that
there is a defined functional specificity for AUUUA motif in
splice-deleted 3⬘ UTR but not the AUUUA in alternatively spliced
3⬘ UTR TCR ␨ mRNA.
8255
8256
REGULATION OF SPLICE-DELETED 3⬘ UTR TCR ␨-CHAIN
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