Stability and Translation of TCR ζ mRNA Are Regulated by the Adenosine-Uridine-Rich Elements in Splice-Deleted 3′ Untranslated Region of ζ-Chain¹²

The 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 ζ-chain with the FcRγ chain (3) and aggregation of lipid rafts on the cell surface membrane (4).

The TCR ζ gene is located in chromosome 1q23.1 (5, 6, 7) an area that has been assigned susceptibility for the development of SLE (8, 9, 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 ζ mRNA and protein expression is significantly reduced in SLE (12, 13, 14, 15, 16). Nucleotide sequence analysis of the TCR ζ 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 ζ-chain mRNA. The alternatively spliced form of TCR ζ 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 ζ mRNA with different nucleotide sequences of the 3′ UTR also have been recently identified in murine T cells (19).

The defective TCR ζ protein expression in SLE T cells inversely correlates with the level of TCR ζ 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 ζ mRNA that are critical for its cellular expression. The regulation of mRNA stability is often mediated by elements within the 3′ UTR (21, 22, 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 ζ 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, 26, 27, 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 ζ 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 ζ 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 ζ mRNA indicates the presence of ARE both in the deleted and the alternatively spliced 3′ UTR. The TCR ζ-chain with splice-deleted 3′ UTR also contain a 31 nucleotide sequence (from 973 to 1003) that is conserved across the 3′ UTR TCR ζ mRNA of several species (19) implying a role in the stability of TCR ζ 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 ζ mRNA contribute to the decreased expression of TCR ζ protein in SLE T cells, we hypothesized that the 562-bp splice-deleted 3′ UTR of TCR ζ 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 ζ-chain. Therefore, the production of TCR ζ mRNA with splice-deleted 3′ UTR represents a molecular mechanism that contributes to translational regulation of decrease expression of TCR ζ-chain in patients with SLE.

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 ζ mAb 6B10.2, recognizing the amino acids 31–45 of the polypeptide (N-terminal mAb), was purchased from BD Pharmingen. The C-terminal TCR ζ 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.

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 ζ 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 ζ 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 ζ-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 ζ 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 ζ-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).

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 μl of Opti-MEM serum-free medium (Invitrogen Life Technologies). Twelve micrograms of expression vector plasmid, pcDNA 3.1 V5 HIS TOPO containing TCR ζ-chain with splice-deleted or alternatively spliced 3′ UTR was added and electroporated at 250 V, 960 μF in a 0.4-cm cuvette (Bio-Rad). Transfected cells were lysed at different time points after incubation with actinomycin D (5 μg/ml) or cycloheximide (10 μg/ml), and the mRNA was isolated after lysis of the cell membrane by Nonidet P-40 (14).

Total RNA was prepared from transfected COS-7 cells and 1 μg 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 ζ mRNA with splice-deleted and alternatively spliced 3′ UTR levels was normalized to the corresponding β-actin.

cDNA encoding for the splice-deleted and alternatively spliced 3′ UTR TCR ζ was obtained by PCR amplication. The cDNA was used as the template for the in vitro transcription of 3′ UTR TCR ζ. The splice-deleted and alternatively spliced TCR ζ-chains were transcribed and translated using TNT T7 quick-coupled rabbit reticulocyte lysate transcription/translational system as recommended by the manufacturer (Promega). Plasmids (8 μg) 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).

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. Wild-type and splice-deleted 3′ UTR TCR ζ point mutant (m) constructs were generated by using the Quik-Change Site Directed Mutagenesis kit (Stratagene) of the TCR ζ 3′ UTR expression vector construct. All three ATTTA sites in the 3′ UTR of TCR ζ cDNA were mutated to GGGTA (resulting in 3′ UTR TCR ζ mARE1, 3′ UTR TCR ζ mARE2, and 3′ UTR TCR ζ mCS). The three ATTTA to GGGT mutations were made at position 636 (resulting in 3′ UTR TCR ζ mARE1), position 705 (resulting in 3′ UTR TCR ζ mARE2), and position 985 (resulting in 3′ UTR TCR ζ mCS).

Equal amounts (20 μg) 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 ζ (clone 6B10.2). The binding was detected using an ECL system (Amersham Biosciences) according to the manufacturer’s instructions.

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 ζ-chain mRNAs were amplified by PCR with XbaI site and cloned into the XbaI 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 luciferase constructs with various sequences of 3′ UTR TCR ζ 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 μl of reporter lysis buffer (Promega). Luciferase activity was assayed with 20 μl of lysate and 80 μl of luciferase assay reagent (Promega) in a TD20/20 luminometer (Turner Designs) using a commercially available kit (Promega).

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.

We recently reported that SLE T cells express high amounts of an alternatively spliced form of TCR ζ mRNA that lacks 562 nt (672–1233) in the 3′ UTR. The resultant TCR ζ mRNA displays decreased stability and translation efficiency and thus contributes to diminished expression of TCR ζ-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 ζ, we cloned the splice-deleted and alternatively spliced 3′ UTR TCR ζ (Fig. 2,A) 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 ζ protein in transfected cells by immunoblotting. As shown in Fig. 2,B, TCR ζ 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 ζ 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-β-actin Ab was used to reblot stripped membranes and confirm equal protein loading. The expression of TCR ζ protein by alternatively spliced 3′ UTR was very low in comparison to splice-deleted form (Fig. 2,B). Densitometric analysis of the immunoblots showed higher amounts of TCR ζ protein expression (>7-fold) in cells transfected with splice-deleted 3′ UTR than alternatively spliced constructs (Fig. 2 C). These results suggest that splice-deleted 3′ UTR of TCR ζ mRNA has a major impact on the expression of TCR ζ protein.

To rule out the possibility that higher production of TCR ζ 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 ζ-chain showed that TCR ζ with alternatively spliced 3′ UTR construct produced significantly lower amounts of protein than the splice-deleted 3′ UTR construct (Fig. 2,D). We confirmed these results by stripping and reprobing the blots by using TCR ζ-specific Ab (Fig. 2,E). Densitometric analysis showed that the level of expression of TCR ζ protein in cells transfected with splice-deleted 3′ UTR resulted in a 10-fold increase than the alternatively spliced 3′ UTR (Fig. 2 F). These results strongly support the presence of regulatory elements within the splice-deleted 562-bp region of 3′ UTR TCR ζ mRNA.

To establish that the decreased stability of TCR ζ 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 ζ mRNA that lacks this 562 bp in transfected COS-7 cells. Transfected cells were incubated with transcription inhibitor actinomycin D (5 μg/ml) for different periods of time (0, 2, 6, and 10 h) and the levels of expression of TCR ζ 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 ζ 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 ζ mRNA expression. These experiments demonstrate that splice-deleted 562-bp (nt 672–1233) segment of TCR ζ mRNA contributes to the stability of the TCR ζ 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 ζ mRNA with splice-deleted and alternatively spliced 3′ UTR in transfected COS-7 cells treated with actinomycin D (5 μg/ml) for 0, 2, 6, or 10 h to confirm the observed differences in the stability of the TCR ζ 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 ζ mRNA (Fig. 4) in Cepheid Thermocycler as described in Materials and Methods. The TCR ζ mRNAs with splice-deleted and alternatively spliced 3′ UTR were evaluated as the relative quantity against β-actin mRNA in the cells before treatment with actinomycin D (data not shown). Real-time 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 ζ 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 ζ mRNA with splice-deleted 3′ UTR was higher and degraded to a lower extent than alternatively spliced 3′ UTR in transfected COS-7 cell.

To determine whether the splice-deleted region that confers the stability to TCR ζ mRNA contributes also to its translation efficiency, we transfected to COS-7 cells with splice-deleted and alternatively spliced 3′ UTR of TCR ζ constructs to examine the levels of expression of TCR ζ protein in transfected cells in the presence of a protein synthesis inhibitor, cycloheximide (10 μg/ml) for different periods of time (0, 2, 6, and 10 h). The levels of expression of 16 kDa TCR ζ protein by splice-deleted and alternatively spliced 3′ UTR were quantified by comparing with β-actin expression (Fig. 5). Although in transfected COS-7 cells, there was no significant decrease in the TCR ζ protein by 2 h for either constructs, by 6 and 10 h we recorded no significant decrease in the level of TCR ζ 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 ζ 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 ζ protein expression with alternatively spliced 3′ UTR was significantly decreased (Fig. 5 C). These experiments indicated that TCR ζ with splice-deleted 3′ UTR restored the translational efficiency of TCR ζ mRNA in transfected COS-7 cells.

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 that of TCR ζ-chain (Fig. 6,A). Luciferase constructs containing TCR ζ-chain with splice-deleted and alternatively spliced 3′ UTR were made by PCR amplification by engineered primers containing XbaI site (Fig. 6,B). 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. 7,A). 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. 6,C). Finally, luciferase clones were reconfirmed by sequence analysis (data not shown). The resulting luciferase reporter constructs containing TCR ζ splice-deleted or alternatively spliced 3′ UTR were individually cotransfected with β-galactosidase to COS-7 cells. As shown in Fig. 6,D, 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. 6 E, 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 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.

There are three of these AREs (position 636, 705, and 985) found in 3′ UTR TCR ζ. 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. 7,A) 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 ζ-chain. Luciferase constructs containing TCR ζ-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. 7,B). 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 ζ were individually cotransfected with β-galactosidase to COS-7, Jurkat cells, and normal T cells. As shown in Fig. 7,C, the luciferase activities in transfected COS-7 cells were significantly decreased with the ARE1 of alternatively spliced 3′ UTR TCR ζ construct compared with ARE2 and CS of splice-deleted 3′ UTR TCR ζ (COS-7; alternatively spliced to ARE2; p = 0.038 and alternatively spliced to CS; p = 0.002). These results were reproduced when Jurkat 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 ζ (Fig. 7 D). 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 ζ mRNA that are absent in alternatively spliced form confer stability to TCR ζ mRNA where as the ARE1 3′ UTR that is only present in alternatively spliced form has no control in regulating the TCR ζ mRNA stability.

To provide further evidence that the ARE present in the splice-deleted 3′ UTR of TCR ζ mRNA have a major impact on the translation regulation of TCR ζ mRNA expression that result in higher production of TCR ζ protein, we performed site-directed mutagenesis. We have transcribed and translated TCR ζ 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 ζ. A schematic representation of 3′ UTR TCR ζ mRNA including splice-deleted and alternatively spliced form and the position of the AUUUA motifs is shown in Fig. 8,A. The sequences of the respective 3′ UTRs are shown in Fig. 1,C. There are three AUUUA motifs in the 3′ UTR of TCRζ 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 ζ protein expression in the transfected cells. The TCR ζ protein expression was studied by transfecting mutant and splice-deleted WT 3′ UTR TCR ζ vectors into COS-7 cells and measuring the expression of TCR ζ protein levels by Western blotting using TCR ζ mAb (Fig. 8,B). The splice-deleted WT 3′ UTR vector was used as a control. Protein expression levels of TCR ζ were affected by the mutation at ARE2 and CS (ARE3) but not at ARE1 of 3′ UTR TCR ζ mRNA. As a control for protein loading, these blots were stripped and reprobed with anti-β-actin Ab and expression of β-actin was noted to be normal in all experiments. Densitometric analysis (Fig. 8 C) showed that the TCR ζ 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 ζ mRNA results in induced down-regulation of TCR ζ 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 ζ mRNA.

In this study, we demonstrate that splice-deleted 562 bp within the 3′ UTR of TCR ζ mRNA is directly involved in the positive regulation of transcription, stability, and translation of TCR ζ 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 ζ mRNA that lacks these critical elements is the predominant form of TCR ζ mRNA observed in SLE T cells. Our results demonstrate that the production of alternatively spliced forms of TCR ζ lacking these critical residues in its 3′ UTR region represents an important molecular mechanism that contributes to reduced expression of TCR ζ-chain mRNA and protein in SLE.

Regulation of mRNA stability is often mediated by elements within the 3′ UTR (21, 22, 23). The 3′ UTR of mRNAs plays an important role in regulating gene expression at the posttranscriptional level (21, 25, 26, 27, 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 proto-oncogenes (40, 41, 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 β-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 trans-activating factors (22, 31).

The TCR ζ with splice-deleted 3′ UTR contains a 31 nucleotide sequence (from 973 to 1003) that is conserved across the TCR ζ 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 ζ mRNA. However, the ARE that is present upstream of the splice-deleted region (position at 636), has no role in the regulation of TCR ζ 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 ζ-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, 52, 53, 54, 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 ζ-chain precisely regulate the expression of TCR ζ protein in T cells is currently under investigation in this laboratory.

In conclusion, we have identified two AREs within the 3′ UTR of TCR ζ 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 ζ 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 ζ protein expression and abnormal T cell function.

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.

The authors have no financial conflict of interest.