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• W2074524976 abstract "The molecular mechanisms involved in the aberrant expression of T cell receptor (TCR) ζ chain of patients with systemic lupus erythematosus are not known. Previously we demonstrated that although normal T cells express high levels of TCR ζ mRNA with wild-type (WT) 3′ untranslated region (3′ UTR), systemic lupus erythematosus T cells display significantly high levels of TCR ζ mRNA with the alternatively spliced (AS) 3′ UTR form, which is derived by splice deletion of nucleotides 672–1233 of the TCR ζ transcript. Here we report that the stability of TCR ζ mRNA with an AS 3′ UTR is low compared with TCR ζ mRNA with WT 3′ UTR. AS 3′ UTR, but not WT 3′ UTR, conferred similar instability to the luciferase gene. Immunoblotting of cell lysates derived from transfected COS-7 cells demonstrated that TCR ζ with AS 3′ UTR produced low amounts of 16-kDa protein. In vitro transcription and translation also produced low amounts of protein from TCR ζ with AS 3′ UTR. Taken together our findings suggest that nucleotides 672–1233 bp of TCR ζ 3′ UTR play a critical role in its stability and also have elements required for the translational regulation of TCR ζ chain expression in human T cells. The molecular mechanisms involved in the aberrant expression of T cell receptor (TCR) ζ chain of patients with systemic lupus erythematosus are not known. Previously we demonstrated that although normal T cells express high levels of TCR ζ mRNA with wild-type (WT) 3′ untranslated region (3′ UTR), systemic lupus erythematosus T cells display significantly high levels of TCR ζ mRNA with the alternatively spliced (AS) 3′ UTR form, which is derived by splice deletion of nucleotides 672–1233 of the TCR ζ transcript. Here we report that the stability of TCR ζ mRNA with an AS 3′ UTR is low compared with TCR ζ mRNA with WT 3′ UTR. AS 3′ UTR, but not WT 3′ UTR, conferred similar instability to the luciferase gene. Immunoblotting of cell lysates derived from transfected COS-7 cells demonstrated that TCR ζ with AS 3′ UTR produced low amounts of 16-kDa protein. In vitro transcription and translation also produced low amounts of protein from TCR ζ with AS 3′ UTR. Taken together our findings suggest that nucleotides 672–1233 bp of TCR ζ 3′ UTR play a critical role in its stability and also have elements required for the translational regulation of TCR ζ chain expression in human T cells. The TCR ζ chain is a component of the T cell receptor (TCR) 1The abbreviations used are: TCR, T cell receptor; SLE, systemic lupus erythematosus; AS, alternatively spliced; UTR, untranslated region; mAb, monoclonal antibody; RT-PCR, reverse transcription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild-type.1The abbreviations used are: TCR, T cell receptor; SLE, systemic lupus erythematosus; AS, alternatively spliced; UTR, untranslated region; mAb, monoclonal antibody; RT-PCR, reverse transcription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild-type. complex that plays a critical role in the assembly and transport of the TCR complex to the cell surface and signal transduction through the TCR that leads to T cell activation. The TCR ζ gene is located in chromosome 1q23.1 (1Jensen J.P. Bates P.W. Yang M. Vierstra R.D. Weissman A.M. J. Biol. Chem. 1995; 270: 30408-30414Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 2Weissman A.M. Hou D. Orloff D.G. Modi W.S. Seuanez H. O'Brien S.J. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9709-9713Crossref PubMed Scopus (158) Google Scholar, 3Stacey M. Barlow A. Hulten M. Chromosome Res. 1997; 5: 279Crossref PubMed Google Scholar), and genome-wide screening for systemic lupus erythematosus (SLE) susceptibility genes demonstrated a linkage between chromosome 1 and SLE susceptibility (4Moser K.L. Neas B.R. Salmon J.E. Yu H. Gray-McGuire C. Asundi N. Bruner G.R. Fox J. Kelly J. Henshall S. Bacino D. Dietz M. Hogue R. Koelsch G. Nightingale L. Shaver T. Abdou N.I. Albert D.A. Carson C. Petri M. Treadwell E.L. James J.A. Harley J.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14869-14874Crossref PubMed Scopus (416) Google Scholar, 5Gaffney P.M. Kearns G.M. Shark K.B. Ortmann W.A. Selby S.A. Malmgren M.L. Rohlf K.E. Ockenden T.C. Messner R.P. King R.A. Rich S.S. Behrens T.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14875-14879Crossref PubMed Scopus (325) Google Scholar, 6Shai R. Quismorio Jr., F.P. Li L. Kwon O.J. Morrison J. Wallace D.J. Neuwelt C.M. Brautbar C. Gauderman W.J. Jacob C.O. Hum. Mol. Genet. 1999; 8: 639-644Crossref PubMed Scopus (280) Google Scholar). SLE T cells express decreased amounts of TCR ζ mRNA and protein (7Liossis S.N. Ding D.Z. Dennis G.J. Tsokos G.C. J. Clin. Investig. 1998; 101: 1448-1457Crossref PubMed Scopus (290) Google Scholar, 8Takeuchi T. Tsuzaka K. Pang M. Amano K. Koide J. Abe T. Int. Immunol. 1998; 10: 911-921Crossref PubMed Scopus (67) Google Scholar, 9Brundula V. Rivas L.J. Blasini A.M. Paris M. Salazar S. Stekman I.L. Rodriguez M.A. Arthritis Rheum. 1999; 42: 1908-1916Crossref PubMed Scopus (80) Google Scholar). However, the TCR/CD3-initiated signaling events are increased in intensity compared with those in normal T cells (10Tsokos G.C. Nambiar M.P. Tenbrock K. Juang Y.T. Trends Immunol. 2003; 24: 259-263Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Specifically, cross-linking of the CD3-TCR complex in SLE T cells leads to increased free intracytoplasmic calcium levels (11Vassilopoulos D. Kovacs B. Tsokos G.C. J. Immunol. 1995; 155: 2269-2281PubMed Google Scholar) and tyrosine phosphorylation of cytosolic proteins (7Liossis S.N. Ding D.Z. Dennis G.J. Tsokos G.C. J. Clin. Investig. 1998; 101: 1448-1457Crossref PubMed Scopus (290) Google Scholar). This “overexcitability” of the SLE T cell has been attributed to the fact that the missing TCR ζ chain is replaced by the FcRγ chain (12Enyedy E.J. Nambiar M.P. Liossis S.N. Dennis G. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: 1114-1121Crossref PubMed Scopus (150) Google Scholar) and the surface membrane lipid rafts are aggregated (13Krishnan S. Nambiar M.P. Warke V.G. Fisher C.U. Mitchell J. Delaney N. Tsokos G.C. J. Immunol. 2004; 172: 7821-7831Crossref PubMed Scopus (151) Google Scholar). Because replenishment of the TCR ζ chain in SLE T cells corrects signaling aberrations and increases interleukin-2 production (14Nambiar M.P. Fisher C.U. Warke V.G. Krishnan S. Mitchell J.P. Delaney N. Tsokos G.C. Arthritis Rheum. 2003; 48: 1948-1955Crossref PubMed Scopus (95) Google Scholar), it is clear that the decreased TCR ζ chain expression is central in the disease process. The molecular mechanisms of diminished expression of the TCR ζ mRNA and protein in SLE T cells remain unknown. The TCR ζ gene 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 to more than 8 kb (15Pang M. Abe T. Fujihara T. Mori S. Tsuzaka K. Amano K. Koide J. Takeuchi T. Arthritis Rheum. 1998; 41: 1456-1463Crossref PubMed Scopus (27) Google Scholar, 16Jensen J.P. Hou D. Ramsburg M. Taylor A. Dean M. Weissman A.M. J. Immunol. 1992; 148: 2563-2571PubMed Google Scholar). Nucleotide sequence analysis of the TCR ζ gene showed increased frequency of alternatively spliced (AS) forms missing various exons as well as splice insertion in SLE T cells (17Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Wong H.K. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: 1336-1350Crossref PubMed Scopus (66) Google Scholar). Analysis of the 3′ untranslated region (UTR) showed a novel 344-bp AS form with a deletion of nucleotides from 672 to 1233 of exon VIII of the TCR ζ mRNA. Using a specific primer that spans either side of the newly identified alternatively spliced site, we recently reported that the AS form of TCR ζ mRNA with 344 bp 3′ UTR was predominantly expressed in SLE T cells compared with normal T cells (14Nambiar M.P. Fisher C.U. Warke V.G. Krishnan S. Mitchell J.P. Delaney N. Tsokos G.C. Arthritis Rheum. 2003; 48: 1948-1955Crossref PubMed Scopus (95) Google Scholar). In SLE T cells, defective TCR ζ protein expression inversely correlates with the level of TCR ζ mRNA with AS 3′ UTR (18Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. Autoimmun. 2001; 16: 133-142Crossref PubMed Scopus (71) Google Scholar) and directly with WT 3′ UTR. Thus, a molecular switching of TCR ζ mRNA with WT 3′ UTR to AS 3′ UTR in T cells contributes to the regulation of TCR ζ chain expression. Several AS isoforms of the TCR ζ mRNA with different nucleotide sequences of the 3′ UTR have been recently identified in murine T cells (19Nocentini G. Ronchetti S. Bartoli A. Testa G. D'Adamio F. Riccardi C. Migliorati G. Eur. J. Immunol. 1995; 25: 1405-1409Crossref PubMed Scopus (14) Google Scholar). The expression of these TCR ζ mRNA isoforms with alternatively spliced 3′ UTR can be modulated by exposure to pharmacologic agents (20Giunchi L. Nocentini G. Ronchetti S. Bartoli A. Riccardi C. Migliorati G. Mol. Cell. Biochem. 1999; 195: 47-53Crossref PubMed Scopus (6) Google Scholar, 21Ronchetti S. Nocentini G. Giunchi L. Bartoli A. Moraca R. Riccardi C. Migliorati G. Cell. Immunol. 1997; 178: 124-131Crossref PubMed Scopus (8) Google Scholar). The regulation of mRNA decay rate is an important control point in determining the abundance of cellular transcripts (22Conne B. Stutz A. Vassalli J.D. Nat. Med. 2000; 6: 637-641Crossref PubMed Scopus (460) Google Scholar). The 3′ UTRs of eukaryotic mRNAs are known to play a crucial role in post-transcriptional regulation of gene expression by modulating nucleocytoplasmic mRNA transport, polyadenylation status, subcellular targeting, translation efficiency, stability, and rates of degradation (23Donnini M. Lapucci A. Papucci L. Witort E. Jacquier A. Brewer G. Nicolin A. Capaccioli S. Schiavone N. J. Biol. Chem. 2004; 279: 20154-20166Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 24Nishimori T. Inoue H. Hirata Y. Life Sci. 2004; 74: 2505-2513Crossref PubMed Scopus (12) Google Scholar, 25Tsuzaka K. Fukuhara I. Setoyama Y. Yoshimoto K. Suzuki K. Abe T. Takeuchi T. J. Immunol. 2003; 171: 2496-2503Crossref PubMed Scopus (52) Google Scholar). Selective expression of TCR ζ mRNA with very short AS 3′ UTR in SLE suggests that it plays an important role in the down-regulation of TCR ζ chain expression in SLE T cells. Therefore, we conducted studies to determine the stability of TCR ζ mRNA with an AS 3′ UTR in SLE T cells and the transport and subsequent translation of TCR ζ mRNA with AS 3′ UTR in normal and SLE T cells. In this report we demonstrate that AS 3′ UTR confers instability to the TCR ζ mRNA, as it does to the mRNA of other genes, and thus contributes to decreased levels of TCR ζ mRNA. In addition, we show that the TCR ζ mRNA with AS 3′ UTR displayed poor translation efficiency leading to the production of low amounts of protein. Therefore, the production of TCR ζ mRNA with short 3′ UTR represents a molecular mechanism that contributes to decreased expression of TCR ζ chain in SLE T cells. SLE Subjects and Controls—Patients fulfilling the American College of Rheumatology Classification criteria for SLE (26Tan E.M. Cohen A.S. Fries J.F. Masi A.T. McShane D.J. Rothfield N.F. Schaller J.G. Talal N. Winchester R.J. Arthritis Rheum. 1982; 25: 1271-1277Crossref PubMed Scopus (12339) Google Scholar) were chosen for the study. Subjects who were on prednisone were asked not to take this medication at least 24 h before drawing the blood. Disease activity for the SLE patients was scored by the SLE disease activity index (SLEDAI) system. Our studies included patients with SLEDAI scores ranging from 0 to 20. The protocol of the study was approved by the Human Use Committees of the involved institutes. Written informed consent was obtained from all participating subjects. Cells and Antibodies—Peripheral blood mononuclear cells from heparinized venous blood were obtained by density gradient centrifugation over Ficoll. T cells were isolated by magnetic separation of non-T cells using a mixture of hapten-conjugated antibodies and MACS microbeads coupled to anti-hapten mAb as described earlier (17Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Wong H.K. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: 1336-1350Crossref PubMed Scopus (66) Google Scholar) (Miltenyi Biotech, Auburn, CA). In all cases, the percentage of T cells in the isolated subpopulation was >97% as determined by fluorescence-activated cell sorter analysis. The TCR ζ chain mAb 6B10.2 (Santa Cruz Biotechnology, Santa Cruz, CA) recognizing amino acids 31–45 of the polypeptide (N-terminal mAb) was purchased from BD Biosciences. The C-terminal TCR ζ chain mAb recognizing amino acids 145–161 (28Hall C.G. Sancho J. Terhorst C. Science. 1993; 261: 915-918Crossref PubMed Scopus (63) Google Scholar) and horseradish peroxidase-conjugated anti-phosphotyrosine mAb 4G10 were purchased from Upstate Biotechnology (Lake Placid, NY). CD3-PE was from Sigma. OKT3 was from Orthoclone Biotech (Raritan, NJ). CD4-PE, CD8-PE, and CD14-PE were from Immunotech (Coulter Corp., Miami, FL), CD16-PE was from BD Biosciences. Fluorescent isotype controls were purchased either from Sigma, Jackson ImmunoResearch Laboratories (West Grove, PA), or BD Biosciences. RT-PCR of TCR ζ mRNA with Wild-type and Alternatively Spliced 3′ Untranslated Region—Total RNA was isolated using RNeasy mini kit (Qiagen) from 5 million T cells from SLE or normal volunteers according to the supplier's directions. Single-stranded cDNA was synthesized by using the AMV reverse transcriptase-based reverse transcription system from Promega (Madison, WI) and oligo(dT) primer as instructed by the manufacturer. The primers for the amplification were synthesized by Sigma-Genosys (The Woodlands, TX). The 5′ primer for the PCR amplification of WT and AS TCR ζ mRNA was 5′-AGC CTC TGC CTC CCA GCC TCT TTC TGA G-3′ (sense bp 34–62 according to the numbering of Weissman et al.; Ref. 2Weissman A.M. Hou D. Orloff D.G. Modi W.S. Seuanez H. O'Brien S.J. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9709-9713Crossref PubMed Scopus (158) Google Scholar) (Sigma-Genosys). The 3′ primer for the specific amplification of TCR ζ mRNA with WT 3′ UTR was 5′-CAA CAG TCT GTG TGT GAA GGT TTG GAG-3′ (antisense bp 737–711). This primer is located in the splice-deleted region of the TCR ζ mRNA (Fig. 1) and is absent in TCR ζ mRNA with AS 3′ UTR. TCR ζ mRNA with AS 3′ UTR was specifically amplified by a primer that spans both sides of the AS site, making it non-complementary, and will not anneal with the WT TCR ζ mRNA. The sequence of this primer is 5′-CAT CTT CTG GCC CTT CAG TGG CTG AGA AGA GTG AA-3′ (antisense bp 1235–1234 and 671–649). β-actin was used as a control, and the primers were forward, 5′-CAT GGG TCA GAA GGA TTC CT-3′, and reverse, 5′-AGC TGG TAG CTC TTC TCC A-3′. The reverse transcription product was diluted 2- to 5-fold and used for PCR amplification of TCR ζ mRNA or β-actin control. The amplification was carried out with a high fidelity PCR system from Roche Applied Science in a Biometra T-3 thermal cycler after initial denaturation at 94 °C for 4 min, 33 cycles at 94 °C, 45 s; 67 °C, 1 min; 72 °C for 2 min; and a final extension at 72 °C for 7 min. 10–15 μl of the PCR products were electrophoresed on 1.2–1.5% SeaKem-agarose gel (FMC BioProducts, Rockland, ME) and visualized with ethidium bromide or SYBR green staining. Quantitation of the RT-PCR product was done using the software GEL-PRO (Media Cybernetics, Silver Spring, MD). Real-time Quantitative PCR of TCR ζ mRNA with Wild-type and Alternatively Spliced 3′ UTR—Real-time quantitative PCR was carried out with a Cepheid Smart Thermocycler by adding SYBR green to the reaction mixture. The SYBR green-based real-time quantitative PCR technique was used to detect the expression of TCR ζ mRNA with WT and AS 3′ UTR. PCR beads were used for amplification (Amersham Biosciences), and the amplification was carried out in 25-μl tubes. Total RNA was prepared from normal and SLE T cells, and 1 μg of RNA was reversed transcribed into cDNA and diluted 10-fold for real-time quantitative PCR. The PCR mixture consists of 2 μl of diluted cDNA, 5 μl of SYBR green diluted (1:500) containing PCR master mixture, and 150 μm each primer in a total volume 25 μl. The specific primers for real-time quantitative PCR were designed as above by Sigma-Genosys. Real-time quantitative PCR was carried out with a Cepheid Smart Thermocycler by adding SYBR green (1:500 dilution) to the reaction mixture. The condition for real-time quantitative PCR was initial denaturation of 94 °C for 60 s followed by cycles of 94 °C for 15 s, 67 °C for 15 s, and 72 °C for 30 s for 45 cycles, followed by a melting point determination that results in a single peak if the amplification is specific. Real-time quantitative PCR products were also separated on a 1.5% agarose gel to visualize the formation of correct PCR products. The expression level for each gene was shown by the cycle numbers needed for the cDNA to be amplified to reach a threshold. The cycle numbers were also converted into arbitrary amounts of DNA using standard curves generated for each pair of primers, and the results were normalized to the housekeeping gene GAPDH mRNA in the same sample. The cycle threshold value was generated by ABI PRISM 7700 SDS software version 1.7 and then exported to an Excel spreadsheet where equations from the standard curve were generated. Using the cycle threshold values, concentrations of TCR ζ mRNA with WT and AS 3′ UTR as well as GAPDH mRNAs were calculated from the equations. 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 WT and AS 3′ UTR levels were normalized to the corresponding GAPDH. PCR Amplification, Cloning, and Expression of the Wild-type and Alternatively Spliced TCR ζ mRNA from T Cells—cDNA was synthesized from total RNA by oligo(dT) primer and AMV reverse transcriptase as described above. The full-length TCR ζ mRNA with WT and AS 3′ UTR was amplified by PCR using primers 5′-AGC CTC TGC CTC CCA GCC TCT TTC TGA G-3′ (sense bp 34–62) and 5′-CCC TAG TAC ATT GAC GGG TTT TTC CTG-3′ (antisense bp 1472–1446). The amplification was carried out using a high fidelity PCR system from Roche Applied Science in a Biometra T-3 thermal cycler after initial denaturation at 94 °C for 6 min, 33 cycles at 94 °C, 1 min; 67 °C, 1 min; 72 °C for 2 min, and a final extension at 72 °C for 7 min. The PCR products containing 1438 bp WT and 916 bp AS TCR ζ were ligated to unidirectional pcDNA 3.1 His TOPO vector (Invitrogen). Minipreps were prepared from 12 recombinant clones, and the direction of the insert was verified by restriction mapping using BamHI 1. Wild-type and AS TCR ζ clones with proper orientation were subjected to DNA sequencing from both orientations on an ABI 377 sequencer using the ABI dye terminator cycle sequencing kit (ABI PRISM; Applied Biosystems Inc., Foster City, CA). Transfection of TCR ζ with WT and AS 3′ UTR to COS-7 cells—The day before transfection, COS-7 cells were subcultured in RPMI 1640 containing 10% fetal bovine serum and penicillin streptomycin at 37 °C ina5%CO2 incubator. For transfection, cells were trypsinized, washed, and resuspended in 250 μl of Opti-MEM serum-free medium (Invitrogen). 15 μg of plasmid pcDNA 3.1 V5 HIS TOPO containing TCR ζ with WT or AS 3′ UTR was added and electroporated at 250 V, 960 μF in a 0.4-cm cuvette (Bio-Rad). Electroporated cells were immediately washed in medium and cultured. Transfected cells were lysed at different time points after incubation with actinomycin D (5 μg/ml), and the mRNA was isolated after lysis of the cell membrane by Nonidet P-40 (7Liossis S.N. Ding D.Z. Dennis G.J. Tsokos G.C. J. Clin. Investig. 1998; 101: 1448-1457Crossref PubMed Scopus (290) Google Scholar). In Vitro Transcription and Translation—The WT and AS TCR ζ mRNA were transcribed and translated using the TNT T7 quick-coupled rabbit reticulocyte lysate transcription/translational system as recommended by the manufacturer (Promega). 10 μg of plasmids containing WT or AS TCR ζ was incubated with the transcription/translation system in the presence of Transcend Botin-Lysyl-tRNA for 60 min at 30 °C. The translated product was electrophoresed and transferred to polyvinylidene difluoride membranes. The incorporated biotinylated lysine was detected non-radioactively by blotting with streptavidin-horseradish peroxidase and developed using an ECL chemiluminescent kit (Amersham Biosciences). Reporter Gene Construction and Transient Transfection—The full-length 3′ UTRs of WT and AS of TCR ζ mRNA were amplified by PCR and cloned into the XbaI site downstream of the luciferase reporter gene in the pGL3-Basic and Enhancer vector(s) (Promega). Transcription from this construct is driven by an SV40 promoter. The correct orientations of the clones were verified by restriction mapping and sequencing. Plasmid DNA transfections of Jurkat cells, COS-7 cells, and T cells were carried out in 24-well plates (Corning Inc., Corning, NY) using Lipofectamine™ 2000 reagent (Invitrogen) following the manufacturer's protocol. The day before transfection, 6 × 104 COS-7 cells or 0.6 × 106 Jurkat cells were plated in 0.5 ml of medium/well. For each well, Lipofectamine™ 2000 reagent (2–3 μl) was mixed with plasmid DNA (1.5 μg) in serum-free Opti-MEM to allow DNA-Lipofectamine™ reagent complexes to form. The complexes were added to respective wells and mixed by gently rocking the plate back and forth. The transfected cells were washed with 1× phosphate-buffered saline three times. Luciferase Activity Assays—Luciferase activity was determined using a luciferase assay system (Promega) following the manufacturer's protocol. Briefly, the transfected cells were incubated in a CO2 incubator at 37 °C for 18 h and then lysed with 60 μl of reporter lysis buffer (Promega). Cellular debris was removed by centrifugation for 2 min at 12,000 rpm. 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). Light output was measured over a 10-s time period in triplicate for each sample. Relative luciferase activity was calculated by averaging the readings. Transfection efficiency was established in all samples by cotransfection with 1 μg of a plasmid encoding the cytomegalovirus promoter-driven β-galactosidase gene. Luciferase activity was normalized for transfection efficiency using the corresponding β-galactosidase activity. The data are presented as -fold increase in luciferase activity compared with that obtained from cells transfected with vector alone. Densitometry and Statistical Analysis—Densitometry analysis of the Western blot was performed with the software program GelPro (Media Cybernetics). Statistical analysis was done using the software Minitab Version 13 (Minitab Inc., State College, PA) by applying the Student's t test. Stability of TCR ζ mRNA with AS 3′ UTR in SLE T Cells— Cloning and sequence analysis of TCR ζ mRNA revealed the presence of a novel TCR ζ chain transcript with alternatively spliced 3′ UTR in human T lymphocytes (Fig. 1). Although the TCR ζ mRNA with AS 3′ UTR was detected at low levels in normal human T cells, it was found to be predominantly expressed in SLE T cells (18Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. Autoimmun. 2001; 16: 133-142Crossref PubMed Scopus (71) Google Scholar). Because a vast majority of lupus patients display decreased expression of TCR ζ mRNA (7Liossis S.N. Ding D.Z. Dennis G.J. Tsokos G.C. J. Clin. Investig. 1998; 101: 1448-1457Crossref PubMed Scopus (290) Google Scholar, 17Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Wong H.K. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: 1336-1350Crossref PubMed Scopus (66) Google Scholar), we considered the TCR ζ mRNA with AS 3′ UTR to be more unstable than the TCR ζ mRNA with WT 3′ UTR. Accordingly, purified T cells were incubated with transcription inhibitor actinomycin D (5 μg/ml) for different periods of time (0, 1, 2, and 4 h), and the levels of expression of WT and AS 3′ UTR TCR ζ mRNA were quantitated by semiquantitative RT-PCR using specific primers as described under “Materials and Methods.” By calculating the quantity of mRNA as a percentage of the amount at 0 h, we determined that the levels of TCR ζ mRNA with AS 3′ UTR were reduced compared with the WT ζ mRNA in SLE T cells (Fig. 2, A and B). We also compared the stability of the TCR ζ mRNA with WT 3′ UTR to that with the AS 3′ UTR after calculating the ratio of the levels of each mRNA to β-actin mRNA. As shown in Fig. 2C, the stability of AS TCR ζ mRNA is decreased compared with the WT 3′ UTR in SLE T cells (Fig. 2C). Stability of TCR ζ mRNA with AS 3′ UTR in Normal T Cells—To address the question whether the decreased half-life of TCR ζ mRNA with AS 3′ UTR is restricted to SLE T cells, we analyzed the stability of TCR ζ mRNA in normal T cells. Purified normal T cells were incubated with transcription inhibitor actinomycin D (5 μg/ml) for different periods of time, and the levels of TCR ζ mRNA expression with WT and AS 3′ UTR were measured in parallel. We observed that the mRNA stability of TCR ζ mRNA with AS 3′ UTR was also low in normal T cells (Fig. 3, A and B). Although the stability of TCR ζ mRNA with AS 3′ UTR was low in normal cells, it was not as prominent as it was recorded in SLE T cells. After 4 h of incubation with actinomycin D, the amount of TCR ζ mRNA with AS 3′ UTR was 17 and 33% in SLE and normal T cells, respectively (Figs. 2B and 3B). However, the rate of degradation of TCR ζ mRNA with WT 3′ UTR was not significantly different between SLE and normal T cells. Real-time Quantitative PCR Analysis of TCR ζ mRNA with AS 3′ UTR in SLE and Normal T Cells—We used a quantitative real-time RT-PCR to measure the stability of TCR ζ mRNA with WT and AS 3′ UTR in SLE T cells and normal T cells treated with actinomycin D (5 μg/ml) for 0, 1, 2, or 4 h to confirm the observed differences in the stability of the TCR ζ mRNA with WT or AS 3′ UTR. The mRNA from the samples were converted to cDNA by reverse transcriptase. The reverse transcription product of cDNA was PCR amplified using TCR ζ WT- and AS 3′ UTR-specific primers in the presence of SYBR green in a Cepheid thermocycler as described under “Materials and Methods.” To validate the real-time quantitative PCR, the standard curves for TCR ζ mRNA with WT and AS 3′ UTR and GAPDH cDNA were constructed first (data not shown). The cycle threshold values for TCR ζ mRNA with WT/AS 3′ UTR and GAPDH mRNA in the sample were measured in triplicate by real-time quantitative PCR, and the amounts of these mRNA were determined from the standard curves. TCR ζ mRNA with WT or AS 3′ UTR was evaluated as the relative quantity against GAPDH mRNA in the cells before treatment with actinomycin D (Fig. 4). Real-time quantitative PCR demonstrated that WT 3′ UTR mRNA was more stable than AS 3′ UTR mRNA during the first 4 h. These results also show that TCR ζ mRNA with AS 3′ UTR (AS mRNA: GAPDH mRNA = 0 h, p = 0.037; 4 h, p = 0.005) in SLE degraded to a greater extent compared with WT 3′ UTR (WT mRNA: GAPDH mRNA = 0 h, p = 0.017; 4 h, p = 0.009) after 4 h of incubation with actinomycin D in SLE T cells (WT 3′ UTR, p = 0.042 and AS 3′ UTR, p = 0.007) (Fig. 4A). The stability of TCR ζ mRNA with WT 3′ UTR (WT mRNA: GAPDH mRNA = 0 h, p = 0.06; 4h, p = 0.03) and AS 3′ UTR (AS mRNA: GAPDH mRNA = 0h, p = 0.005; 4 h, p = 0.001) was also analyzed in normal T cell by real-time quantitative PCR (WT 3′ UTR, p = 0.05 and AS 3′ UTR, p = 0.015) (Fig. 4B). The results show that TCR ζ mRNA with WT 3′ UTR degraded to a lower extent compared with the TCR ζ mRNA with AS 3′ UTR after 4 h of incubation with actinomycin D (Fig. 4). Stability of TCR ζ mRNA with AS 3′ UTR in COS-7 Cells— Next we studied the stability of TCR ζ mRNA with WT and AS 3′ UTR transfected in COS-7 cells. TCR ζ with WT or AS 3′ UTR was cloned into a eukaryotic expression vector, pcDNA3.1/V5-HIS-TOPO, and transfected to COS-7 cells. After 18 h of transfection, the cells were incubated with actinomycin D for 0 or 4 h, and the mRNA were measured by RT-PCR analysis. Although in COS-7 cells there was no significant degradation of TCR ζ mRNA by 4 h, by 8 h we recorded a significant decrease in the level of TCR ζ mRNA with AS 3′ UTR compared with WT 3′ UTR (Fig. 5). To rule out that the difference was due to differences in export from nucleus or transport of TCR ζ mRNA with WT and AS 3′ UTR from nucleus to cytoplasm as a result of alternative splicing, the transfected cells were lysed at different time points after incubation with actinomycin D and mRNA was isolated after lysis of the cell membrane by Nonidet P-40. Levels of TCR ζ mRNA with WT and AS 3′ UTR were measured by semiquantitative RT-PCR. The data showed that the ratio of TCR ζ mRNA between the cytoplasm and nucleus was similar at different points, suggesting that alternative splicing of TCR ζ 3′ UTR does not affect the transport of the TCR ζ mRNA (data not shown). Translational Regulation of TCR ζ Expression in COS-7 Cells Transfected with TCR ζ Carrying WT and AS 3′ UTR—Our previous data showed that the down-regulation of TCR ζ protein in SLE T cells inversely correlated with the level of TCR ζ mRNA with AS 3′ UTR (18Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. Autoimmun. 2001; 16: 133-142Crossref PubMed Scopus (71) Google Scholar). This finding suggested that, although the coding region is intact in TCR ζ mRNA with AS 3′ UTR, the transl" @default.
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• W2074524976 date "2005-05-01" @default.
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• W2074524976 title "Decreased Stability and Translation of T Cell Receptor ζ mRNA with an Alternatively Spliced 3′-Untranslated Region Contribute to ζ Chain Down-regulation in Patients with Systemic Lupus Erythematosus" @default.
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• W2074524976 doi "https://doi.org/10.1074/jbc.m501048200" @default.
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