Are Regulated by the Adenosine-Uridine-Rich Elements in. -Chain. This information is current as of February 23, 2013.

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of February 23, 2013.

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-Chain

Splice-Deleted 3

Untranslated Region of

ζ

Adenosine-Uridine-Rich Elements in

Are Regulated by the

mRNA

ζ

Stability and Translation of TCR

Madhusoodana P. Nambiar and George C. Tsokos

Tsokos, James W. Robertson, Carolyn U. Fisher,

Bhabadeb Chowdhury, Sandeep Krishnan, Christos G.

http://www.jimmunol.org/content/177/11/8248

2006; 177:8248-8257; ;

J Immunol

References

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, 32 of which you can access for free at:

cites 58 articles

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Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Immunologists All rights reserved.

Copyright © 2006 by The American Association of

9650 Rockville Pike, Bethesda, MD 20814-3994.

The American Association of Immunologists, Inc.,

is published twice each month by

The Journal of Immunology

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Stability and Translation of TCR

mRNA Are Regulated

by the Adenosine-Uridine-Rich Elements in Splice-Deleted

3

Untranslated Region of

-Chain

1,2

Bhabadeb Chowdhury,

3

Sandeep Krishnan,

3

Christos G. Tsokos, James W. Robertson,

Carolyn U. Fisher, Madhusoodana P. Nambiar, and George C. Tsokos

4

Systemic lupus erythematosus (SLE) T cells display reduced expression of TCRprotein. Recently, we reported that in SLE T cells, the residual TCRprotein is predominantly derived from an alternatively spliced form that undergoes splice deletion of 562 nt (from 672 to 1233 bases) within the 3untranslated region (UTR) of TCRmRNA. The stability and translation of the alternatively spliced form of TCRmRNA are low compared with that of the wild-type TCRmRNA. We report that two adenosine-uridine-rich sequence elements (AREs), defined by the splice-deleted 3UTR region, but not an ARE located upstream are responsible for securing TCRmRNA 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 3UTR, which regulate the transcription, mRNA stability, and translation of TCRmRNA. 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.

T

he complex autoimmune disease systemic lupus erythem-atosus (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 mem-brane (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

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 reg-ulatory 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 insta-bility to other genes (24). This effect was not cell type specific, suggesting thattransfactors 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 mod-ulating nucleocytoplasmic mRNA transport, polyadenylation sta-tus, 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 containscis-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

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 markedadvertisementin 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: gtsokos@usa.net

5

Abbreviations used in this paper: SLE, systemic lupus erythematosus; ARE, AU-rich element; WT, wild type; UTR, untranslated region; CS, conserved sequence; m, mutant (in mARE1, mARE2, and mCS).

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contain a 31 nucleotide sequence (from 973 to 1003) that is con-served across the 3⬘ UTR TCR␨mRNA of several species (19) implying a role in the stability of TCR ␨mRNA. Species-con-served 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 al-ternatively spliced 3⬘ UTR TCR ␨mRNA contribute to the de-creased expression of TCR␨protein in SLE T cells, we hypoth-esized that the 562-bp splice-deleted 3⬘ UTR of TCR␨mRNA contains crucialcis-elements that bind factors that may confer sta-bility 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 trans-lational regulation of decrease expression of TCR␨-chain in pa-tients with SLE.

Materials and Methods

Materials, Abs, and cell culture

Unless indicated, all reagents used for biochemical methods were pur-chased from Sigma-Aldrich, Pierce, or Fisher Chemical. Enzymes for re-striction 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 iso-lated 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 3UTR TCRmRNA

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 syn-thesized 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 num-bering 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 ampli-fied by PCR using specific primers designed for splice-deleted and alter-natively 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 withXbaI 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 uni-directional 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 orienta-tions 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 3UTR TCR

The COS-7 cells were subcultured in RPMI 1640 for 24 h before trans-fection containing 10% FBS and penicillin/streptomycin at 37°C in 5% CO2incubator. For transfection, cells were trypsinized, washed, and

re-suspended 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 alterna-tively 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).

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.

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Real-time PCR of TCRmRNA

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 quan-titative PCR. The SYBR Green-based real-time quanquan-titative PCR technique was conducted with Cepheid Smart. The sequences of splice-deleted prim-ers 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.

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 us-ing TNT T7 quick-coupled rabbit reticulocyte lysate transcription/transla-tional system as recommended by the manufacturer (Promega). Plasmids (8

␮g) containing splice-deleted or alternatively spliced 3⬘UTR were incu-bated with transcription/translation system in the presence of Transcend biotin-lysyl-tRNA for 60 min at 30°C. The translated product was electro-phoresed, transferred to polyvinylidene difluoride membranes, and the in-corporated biotinylated lysine was detected nonradioactively by blotting with streptavidin-HRP and developed using ECL chemiluminescent kit (Amersham Biosciences).

Site-directed mutagenesis of splice-deleted 3UTR TCR-chain

Site-directed mutagenesis was performed using the Quik-Change Site Di-rected Mutagenesis kit (Stratagene) according to the manufacturer’s in-structions. Three different mutants were used in the present study. Wild-type and splice-deleted 3⬘UTR TCR␨point mutant (m) constructs were generated by using the Quik-Change Site Directed Mutagenesis kit (Strat-agene) 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).

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 dena-turing and reducing conditions and the proteins transferred onto polyvi-nylidene difluoride membranes (Amersham Biosciences) (36). The immu-noblot analysis was then conducted using specific Abs against TCR ␨ (clone 6B10.2). The binding was detected using an ECL system (Amer-sham 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 withXbaI site and cloned into theXbaI site downstream of the luciferase reporter gene in the pGL3-Basic and enhancer vectors (Promega). The proper orientations of the clones were verified by restriction mapping and sequence analysis. The

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 trans-fected to COS-7 cells by electroporation. The TCR␨ with splice-deleted and alternatively spliced 3⬘ UTR was transcribed and translated using TNT T7 quick-coupled rabbit reticulocyte lysate transcription/transla-tional 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, Densi-tometric 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 polyvinyli-dene difluoride membranes and the incorporated biotin-ylated lysine in expressed protein was detected with streptavidin-HRP and developed using an ECL chemi-luminescent kit.E, Expression of TCR␨protein with splice-deleted 3⬘UTR. The blots were stripped and re-probed 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).

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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) follow-ing the manufacturer’s protocol. Luciferase activity was determined usfollow-ing 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 soft-ware program GEL-PRO (Media Cybernetics). Statistical analyses were performed using the GraphPad Prism version 4.0 software and Minitab version 13.

Results

Expression of TCRprotein by splice-deleted 3UTR TCR

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 de-creased 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 alterna-tively spliced 3⬘UTR TCR␨(Fig. 2A) into a eukaryotic expression vector, pcDNA3.1/V5-HIS-TOPO, and then transfected the con-structs 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 TCRwith splice-deleted 3UTR

To rule out the possibility that higher production of TCR␨protein in cells transfected with the splice-deleted constructs, we per-formed 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 alterna-tively 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 trans-fected 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.

Stability of TCRmRNA with splice-deleted 3UTR TCR

To establish that the decreased stability of TCR ␨ mRNA with alternatively spliced 3⬘UTR was due to the lack of 562-bp splice-deleted 3⬘ UTR in SLE, we examined the stability of TCR ␨ mRNA that lacks this 562 bp in transfected COS-7 cells. Trans-fected cells were incubated with transcription inhibitor actinomy-cin 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 inMaterials 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 ex-pression level of splice-deleted 3⬘ UTR mRNA, whereas at the same time points, we observed a significant reduction of alterna-tively spliced form (6 h, p⫽0.003; 10 h, p⫽0.01) of TCR␨ mRNA expression. These experiments demonstrate that splice-de-leted 562-bp (nt 672–1233) segment of TCR␨mRNA contributes to the stability of the TCR␨mRNA and that the stability of al-ternatively 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 alterna-tively 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

FIGURE 3. Stability of TCR␨mRNA in COS-7 cells transfected with splice-deleted 3⬘UTR by RT-PCR analysis. The TCR␨-chain with splice-deleted 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 inMaterials 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 soft-ware, and the data are presented as a percentage of control. The data shown are representative of three experiments with similar results from three dif-ferent transfections. Error bars indicate SE.

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Thermocycler as described inMaterials 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). Real-time quantitative PCR confirmed our observations that splice-de-leted 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 de-graded to a greater extent compared with splice-deleted 3⬘UTR in transfected cells (Fig. 4). However, these results clearly demon-strate 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 TCRmRNA by splice-deleted 3UTR

To determine whether the splice-deleted region that confers the stability to TCR␨mRNA contributes also to its translation effi-ciency, we transfected to COS-7 cells with splice-deleted and al-ternatively 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 alter-natively spliced 3⬘UTR were quantified by comparing with␤ -ac-tin 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,AandB). 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,

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 trans-fected COS-7 cells.

Stability and expression of luciferase gene by splice-deleted 3UTR 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

FIGURE 4. Stability and expression of TCR␨mRNA with splice-de-leted 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.

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 trans-fection, the cells were incubated with a protein synthesis inhibitor, cyclo-heximide (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␨ pro-tein 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 alter-natively spliced 3⬘UTR TCR␨in transfected COS-7 cells.C, Densito-metric 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).

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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 con-struct 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 lucif-erase 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 ac-tivity 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 splice-deleted 3⬘ UTR but not the alternatively spliced form confers stability to other genes and mediates this effect independent of the type of cell.

Mapping of the stability regions within the splice-deleted 3UTR TCRmRNA

There are three of these AREs (position 636, 705, and 985) found in 3⬘UTR TCR␨. We introduced various sequences con-taining 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 func-tional 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 containingXbaI site and cloned in pGL3-basic and en-hancer vectors immediately after the luciferase gene at the 3⬘ region (Fig. 7B). The luciferase clones were confirmed by re-striction mapping and sequence analysis (data not shown). The resulting luciferase reporter constructs containing ARE1, ARE2, and CS of 3⬘ UTR TCR ␨ were individually cotrans-fected with␤-galactosidase to COS-7, Jurkat cells, and normal T cells. As shown in Fig. 7C, the luciferase activities in trans-fected 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; alter-natively spliced to ARE2;p⫽0.038 and alternatively spliced to CS; p ⫽ 0.002). These results were reproduced when Jurkat

FIGURE 6. Luciferase activity of TCR␨with splice-deleted 3⬘UTR. The splice-deleted and alternatively spliced 3⬘UTR of TCR␨were cloned intoXbaI site downstream of the luciferase gene in the sense orientation and verified by restriction digestion. Luciferase construct containing splice-deleted 3⬘UTR or alternatively spliced (AS) 3⬘UTR was transfected into COS-7 and Jurkat cells.A, Schematic representation of constitutive lucif-erase 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

spliced 3⬘UTR of TCR␨-chain containingXbaI site.C, Verification of luciferase clones by restriction digestion usingXbaI.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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cells (Jurkat; alternatively spliced to ARE2,p⫽0.04; alterna-tively 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 con-fer 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.

FIGURE 7. The role of AU-rich regions in 3⬘UTR TCR␨by reporter gene expression. The various sequences of ARE1-, ARE2-, and CS-containing AREs of 3⬘UTR TCR␨were cloned intoXbaI site down-stream 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 con-stitutive luciferase reporter constructs (not drawn to scale). Transcrip-tion (bent arrow) was driven by the constitutive SV40 promoter up-stream 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 inde-pendent determinations (n⫽3).

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 thetop.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).

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Mutational analyses of splice-deleted 3UTR to identify the specific gene sequences in controlling the translational regulation of TCR-chain

To provide further evidence that the ARE present in the splice-deleted 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 po-sition 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 vec-tors. 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 ex-periments. 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␨ pro-tein 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.

Discussion

In this study, we demonstrate that splice-deleted 562 bp within the 3⬘UTR of TCR␨mRNA is directly involved in the positive reg-ulation 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 alter-natively 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 alter-natively 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 posttranscrip-tional level (21, 25–28). For example, a 171 bp region in the 3⬘ UTR of utrophin mRNA regulates its stability (38, 39). We exam-ined the effect of the presence of the splice-deleted region on the stability and translation of the luciferase reporter mRNA and es-tablished 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

transfactors are not required and the stabilizing effect is simply 3⬘

UTR length-dependent, or the requiredtransfactors 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 proto-oncogenes (40 – 42). Mutation of AUUUA motifs in the 3⬘UTR of IL-3 (43) and c-fosmRNA (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 var-ious mRNA determine stability on instability by binding tran s-activating 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 evi-dence in this study that this conserved region is also involved in the regulation of mRNA stability and expression because at posi-tion 985 it defines a third AUR. Therefore, the splice-deleted re-gion defines two AREs (at positions 705 and 985) that are respon-sible for the stability and sufficient translation of the TCR ␨ mRNA. However, the ARE that is present upstream of the splice-deleted 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 themyc-N and c-fosmRNA in human neuroblas-toma cells results in increased mRNA stability and an aggres-sive clinical behavior of the tumor (49, 50). In contrast, asso-ciation 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 in-stability (51–55). HuR, a nucleocytoplasmic shuttling protein, has also been shown to bind mRNA-defined ARE and contrib-utes 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 in-vestigation 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 func-tional value. Therefore, specific molecular defects in SLE T cells account for decreased TCR ␨protein expression and ab-normal 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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References

1. Theofilopoulos, A. N., and F. J. Dixon. 1981. Etiopathogenesis of murine SLE.

Immunol. Rev.55: 179 –216.

2. Iliopoulos, A. G., and G. C. Tsokos. 1996. Immunopathogenesis and spectrum of infections in systemic lupus erythematosus.Semin. Arthritis Rheum. 25: 318 –336.

3. Tsokos, G. C., M. P. Nambiar, K. Tenbrock, and Y. T. Juang. 2003. Rewiring the T-cell: signaling defects and novel prospects for the treatment of SLE.Trends Immunol.24: 259 –263.

4. Krishnan, S., M. P. Nambiar, V. G. Warke, C. U. Fisher, J. Mitchell, N. Delaney, and G. C. Tsokos. 2004. Alterations in lipid raft composition and dynamics con-tribute to abnormal T cell responses in systemic lupus erythematosus.J. Immunol.

172: 7821–7831.

5. Jensen, J. P., P. W. Bates, M. Yang, R. D. Vierstra, and A. M. Weissman. 1995. Identification of a family of closely related human ubiquitin conjugating en-zymes.J. Biol. Chem.270: 30408 –30414.

6. Weissman, A. M., D. Hou, D. G. Orloff, W. S. Modi, H. Seuanez, S. J. O’Brien, and R. D. Klausner. 1988. Molecular cloning and chromosomal localization of the human T-cell receptor␨chain: distinction from the molecular CD3 complex.

Proc. Natl. Acad. Sci. USA85: 9709 –9713.

7. Stacey, M., A. Barlow, and M. Hulten. 1997. Human T-cell receptor␨chain gene Map position 1q23.1.Chromosome Res.5: 279.

8. Gaffney, P. M., G. M. Kearns, K. B. Shark, W. A. Ortmann, S. A. Selby, M. L. Malmgren, K. E. Rohlf, T. C. Ockenden, R. P. Messner, R. A. King, et al. 1998. A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families. Proc. Natl. Acad. Sci. USA 95: 14875–14879.

9. Moser, K. L., B. R. Neas, J. E. Salmon, H. Yu, C. Gray-McGuire, N. Asundi, G. R. Bruner, J. Fox, J. Kelly, S. Henshall, et al. 1998. Genome scan of human systemic lupus erythematosus: evidence for linkage on chromosome 1q in Afri-can-American pedigrees.Proc. Natl. Acad. Sci. USA95: 14869 –14874. 10. Shai, R., F. P. Quismorio, Jr., L. Li, O. J. Kwon, J. Morrison, D. J. Wallace,

C. M. Neuwelt, C. Brautbar, W. J. Gauderman, and C. O. Jacob. 1999. Genome-wide screen for systemic lupus erythematosus susceptibility genes in multiplex families.Hum. Mol. Genet.8: 639 – 644.

11. Pang, M., T. Abe, T. Fujihara, S. Mori, K. Tsuzaka, K. Amano, J. Koide, and T. Takeuchi. 1998. Up-regulation of␣E␤7, a novel integrin adhesion molecule, on

T cells from systemic lupus erythematosus patients with specific epithelial in-volvement.Arthritis Rheum.41: 1456 –1463.

12. Nambiar, M. P., E. J. Enyedy, C. U. Fisher, S. Krishnan, V. G. Warke, W. R. Gilliland, R. J. Oglesby, and G. C. Tsokos. 2002. Abnormal expression of various molecular forms and distribution of T cell receptor␨chain in patients with systemic lupus erythematosus.Arthritis Rheum.46: 163–174.

13. Nambiar, M. P., E. J. Enyedy, V. G. Warke, S. Krishnan, G. Dennis, H. K. Wong, G. M. Kammer, and G. C. Tsokos. 2001. T cell signaling abnormalities in sys-temic lupus erythematosus are associated with increased mutations/polymor-phisms and splice variants of T cell receptor␨chain messenger RNA.Arthritis Rheum.44: 1336 –1350.

14. Liossis, S. N., D. Z. Ding, G. J. Dennis, and G. C. Tsokos. 1998. Altered pattern of TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus erythematosus: deficient expression of the T-cell receptor␨ chain.J. Clin. Invest.101: 1448 –1457.

15. Takeuchi, T., K. Tsuzaka, M. Pang, K. Amano, J. Koide, and T. Abe. 1998. TCR

␨chain lacking exon 7 in two patients with systemic lupus erythematosus.Int. Immunol.10: 911–921.

16. Brundula, V., L. J. Rivas, A. M. Blasini, M. Paris, S. Salazar, I. L. Stekman, and M. A. Rodriguez. 1999. Diminished levels of T cell receptor␨chains in periph-eral blood T lymphocytes from patients with systemic lupus erythematosus. Ar-thritis Rheum.42: 1908 –1916.

17. Tsuzaka, K., Y. Setoyama, K. Yoshimoto, K. Shiraishi, K. Suzuki, T. Abe, and T. Takeuchi. 2005. A splice variant of the TCR␨mRNA lacking exon 7 leads to the down-regulation of TCR␨, the TCR/CD3 complex, and IL-2 production in systemic lupus erythematosus T cells.J. Immunol.174: 3518 –3525. 18. Nambiar, M. P., C. U. Fisher, V. G. Warke, S. Krishnan, J. P. Mitchell,

N. Delaney, and G. C. Tsokos. 2003. Reconstitution of deficient T cell receptor

␨chain restores T cell signaling and augments T cell receptor/CD3-induced in-terleukin-2 production in patients with systemic lupus erythematosus.Arthritis Rheum.48: 1948 –1955.

19. Nocentini, G., S. Ronchetti, A. Bartoli, G. Testa, F. D’Adamio, C. Riccardi, and G. Migliorati. 1995. T cell receptor␫an alternatively spliced product of the T cell receptor␨gene.Eur. J. Immunol.25: 1405–1409.

20. Nambiar, M. P., E. J. Enyedy, V. G. Warke, S. Krishnan, G. Dennis, G. M. Kammer, and G. C. Tsokos. 2001. Polymorphisms/mutations of TCR-␨ -chain promoter and 3⬘ untranslated region and selective expression of TCR

␨-chain with an alternatively spliced 3⬘untranslated region in patients with sys-temic lupus erythematosus.J. Autoimmun.16: 133–142.

21. Donnini, M., A. Lapucci, L. Papucci, E. Witort, A. Jacquier, G. Brewer, A. Nicolin, S. Capaccioli, and N. Schiavone. 2004. Identification of TINO: a new evolutionarily conservedBCL-2AU-rich element RNA-binding protein.J. Biol. Chem.279: 20154 –20166.

22. Caballero, J. J., M. D. Giro´n, A. M. Vargas, N. Sevillano, M. D. Sua´rez, and R. Salto. 2004. AU-rich elements in the mRNA 3⬘-untranslated region of the rat receptor for advanced glycation end products and their relevance to mRNA sta-bility.Biochem. Biophys. Res. Commun.319: 247–255.

23. Tsuzaka, K., I. Fukuhara, Y. Setoyama, K. Yoshimoto, K. Suzuki, T. Abe, and T. Takeuchi. 2003. TCR␨mRNA with an alternatively spliced 3⬘-untranslated

region detected in systemic lupus erythematosus patients leads to the down-reg-ulation of TCR␨and TCR/CD3 complex.J. Immunol.171: 2496 –2503. 24. Chowdhury, B., C. G. Tsokos, S. Krishnan, J. Robertson, C. U. Fisher,

R. G. Warke, V. G. Warke, M. P. Nambiar, and G. C. Tsokos. 2005. De-creased stability and translation of T cell receptor␨mRNA with an alterna-tively spliced 3⬘-untranslated region contribute to␨chain down-regulation in patients with systemic lupus erythematosus. J. Biol. Chem. 280: 18959 –18966.

25. Schiavone, N., P. Rosini, A. Quattrone, M. Donnini, A. Lapucci, L. Citti, A. Bevilacqua, A. Nicolin, and S. Capaccioli. 2000. A conserved AU-rich ele-ment in the 3⬘untranslated region ofbcl-2 mRNA is endowed with a destabilizing function that is involved inbcl-2 down-regulation during apoptosis.FASEB J.14: 174 –184.

26. Nishimori, T., H. Inoue, and Y. Hirata. 2004. Involvement of the 3⬘-untranslated region of cyclooxygenase-2 gene in its post-transcriptional regulation through the glucocorticoid receptor.Life Sci.74: 2505–2513.

27. Roe, D. F., G. L. Craviso, and J. C. Waymire. 2004. Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3⬘ UTR in a manner that requires transcription.Brain Res. Mol. Brain Res.120: 91–102.

28. Yu, H., S. Stasinopoulos, P. Leedman, and R. L. Medcalf. 2003. Inherent insta-bility of plasminogen activator inhibitor type 2 mRNA is regulated by tristetra-prolin.J. Biol. Chem.278: 13912–13918.

29. Pesole, G., F. Mignone, C. Gissi, G. Grillo, F. Licciulli, and S. Liuni. 2001. Structural and functional features of eukaryotic mRNA untranslated regions.

Gene276: 73– 81.

30. Jensen, L. E., and A. S. Whitehead. 2004. The 3⬘untranslated region of the membrane-bound IL-1R accessory protein mRNA confers tissue-specific desta-bilization.J. Immunol.173: 6248 – 6258.

31. Rydziel, S., A. M. Delany, and E. Canalis. 2004. AU-rich elements in the col-lagenase 3 mRNA mediate stabilization of the transcript by cortisol in osteo-blasts.J. Biol. Chem.279: 5397–5404.

32. Buzby, J. S., S. M. Lee, P. Van Winkle, C. T. DeMaria, G. Brewer, and M. S. Cairo. 1996. Increased granulocyte-macrophage colony-stimulating factor mRNA instability in cord versus adult mononuclear cells is translation-dependent and associated with increased levels of A⫹U-rich element binding factor.Blood

88: 2889 –2897.

33. Wilson, G. M., Y. Sun, J. Sellers, H. Lu, N. Penkar, G. Dillard, and G. Brewer. 1999. Regulation of AUF1 expression via conserved alternatively spliced elements in the 3⬘ untranslated region. Mol. Cell. Biol. 19: 4056 – 4064.

34. Hall, C. G., J. Sancho, and C. Terhorst. 1993. Reconstitution of T cell receptor

␨-mediated calcium mobilization in nonlymphoid cells. Science 261: 915–918.

35. Krishnan, S., J. G. Kiang, C. U. Fisher, M. P. Nambiar, H. T. Nguyen, V. C. Kyttaris, B. Chowdhury, V. Rus, and G. C. Tsokos. 2005. Increased caspase-3 expression and activity contribute to reduced CD3␨expression in systemic lupus erythematosus T cells.J. Immunol.175: 3417–3423.

36. Chowdhury, B., G. Mantile-Selvaggi, L. Miele, E. Cordella-Miele, Z. Zhang, and A. B. Mukherjee. 2002. Lys 43 and Asp 46 in␣-helix 3 of uteroglobin are essential for its phospholipase A2inhibitory activity.Biochem. Biophys. Res.

Commun.295: 877– 883.

37. Warke, V. G., M. P. Nambiar, S. Krishnan, K. Tenbrock, D. A. Geller, N. P. Koritschoner, J. L. Atkins, D. L. Farber, and G. C. Tsokos. 2003. Tran-scriptional activation of the human inducible nitric-oxide synthase promoter by Kru¨ppel-like factor 6.J. Biol. Chem.278: 14812–14819.

38. Gramolini, A. O., G. Belanger, and B. J. Jasmin. 2001. Distinct regions in the 3⬘ untranslated region are responsible for targeting and stabilizing utrophin tran-scripts in skeletal muscle cells.J. Cell Biol.154: 1173–1183.

39. Gramolini, A. O., G. Belanger, J. M. Thompson, J. V. Chakkalakal, and B. J. Jasmin. 2001. Increased expression of utrophin in a slow vs. a fast muscle involves posttranscriptional events.Am. J. Physiol.281: C1300 –C1309. 40. Chen, C. Y., and A. B. Shyu. 1995. AU-rich elements: characterization and

im-portance in mRNA degradation.Trends Biochem. Sci.20: 465– 470. 41. Shaw, G., and R. Kamen. 1986. A conserved AU sequence from the 3⬘

untrans-lated region of GM-CSF mRNA mediates selective mRNA degradation.Cell46: 659 – 667.

42. Sengupta, T. K., S. Bandyopadhyay, D. J. Fernandes, and E. K. Spicer. 2004. Identification of nucleolin as an AU-rich element binding protein involved in

bcl-2mRNA stabilization.J. Biol. Chem.279: 10855–10863.

43. Stoecklin, G., S. Hahn, and C. Moroni. 1994. Functional hierarchy of AUUUA motifs in mediating rapid interleukin-3 mRNA decay. J. Biol. Chem. 269: 28591–28597.

44. Chen, C. Y., and A. B. Shyu. 1994. Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements.Mol. Cell. Biol.14: 8471– 8482.

45. Thiele, A., Y. Nagamine, S. Hauschildt, and H. Clevers. 2006. AU-rich elements and alternative splicing in the␤-catenin 3⬘UTR can influence the human␤ -cate-nin mRNA stability.Exp. Cell Res. 312: 2367–2378.

46. Dean, J. L., G. Sully, A. R. Clark, and J. Saklatvala. 2004. The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase path-way-mediated mRNA stabilisation.Cell. Signal.16: 1113–1121.

47. Cok, S. J., S. J. Acton, A. E. Sexton, and A. R. Morrison. 2004. Identification of RNA-binding proteins in RAW 264.7 cells that recognize a lipopolysaccharide-responsive element in the 3-untranslated region of the murine cyclooxygenase-2 mRNA.J. Biol. Chem.279: 8196 – 8205.

8256 REGULATION OF SPLICE-DELETED 3⬘UTR TCR␨-CHAIN

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48. Cok, S. J., S. J. Acton, and A. R. Morrison. 2003. The proximal region of the 3⬘-untranslated region of cyclooxygenase-2 is recognized by a multimeric protein complex containing HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonu-cleoprotein U.J. Biol. Chem.278: 36157–36162.

49. Adachi, M., R. Watanabe-Fukunaga, and S. Nagata. 1993. Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene oflprmice.Proc. Natl. Acad. Sci. USA90: 1756 –1760. 50. Chagnovich, D., and S. L. Cohn. 1997. Activity of a 40 kDa RNA-binding protein

correlates withMYCNand c-fosmRNA stability in human neuroblastoma.Eur. J. Cancer33: 2064 –2067.

51. Desai-Mehta, A., L. Lu, R. Ramsey-Goldman, and S. K. Datta. 1996. Hyperex-pression of CD40 ligand by B and T cells in human lupus and its role in patho-genic autoantibody production.J. Clin. Invest.97: 2063–2073.

52. Koshy, M., D. Berger, and M. K. Crow. 1996. Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J. Clin. Invest. 98: 826 – 837.

53. Mohan, C., Y. Shi, J. D. Laman, and S. K. Datta. 1995. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis.J. Immunol.

154: 1470 –1480.

54. Rigby, W. F., M. G. Waugh, and B. J. Hamilton. 1999. Characterization of RNA binding proteins associated with CD40 ligand (CD154) mRNA turnover in hu-man T lymphocytes.J. Immunol.163: 4199 – 4206.

55. Hamilton, B. J., A. Genin, R. Q. Cron, and W. F. Rigby. 2003. Delineation of a novel pathway that regulates CD154 (CD40 ligand) expression.Mol. Cell. Biol.

23: 510 –525.

56. Brennan, C. M., I. E. Gallouzi, and J. A. Steitz. 2000. Protein ligands to HuR modulate its interaction with target mRNAs in vivo.J. Cell Biol.151: 1–14. 57. Brennan, C. M., and J. A. Steitz. 2001. HuR and mRNA stability.Cell. Mol. Life

Sci.58: 266 –277.

58. Winzen, R., G. Gowrishankar, F. Bollig, N. Redich, K. Resch, and H. Holtmann. 2004. Distinct domains of AU-rich elements exert different functions in mRNA destabilization and stabilization by p38 mitogen-activated protein kinase or HuR.

Mol. Cell. Biol.24: 4835– 4847.

at Pennsylvania State Univ on February 23, 2013

http://jimmunol.org/

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