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

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. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: PubMed Scopus Google Scholar). of the 3′ untranslated region showed a AS with a deletion of nucleotides from to of of the TCR ζ a that spans of the alternatively spliced we that the AS of TCR ζ mRNA with bp 3′ UTR 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 TCR ζ protein expression with the of TCR ζ mRNA with AS 3′ UTR M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. 2001; PubMed Scopus Google Scholar) and with WT 3′ UTR. a molecular of TCR ζ mRNA with WT 3′ UTR to AS 3′ UTR in T cells to the regulation of TCR ζ chain AS of the TCR ζ mRNA with of the 3′ UTR have been in T cells G. S. A. G. C. G. J. Immunol. 1995; PubMed Scopus Google Scholar). The expression of TCR ζ mRNA with alternatively spliced 3′ UTR by to L. G. S. A. C. G. Mol. 1999; PubMed Scopus Google Scholar, S. G. L. A. R. C. G. Immunol. 1997; PubMed Scopus Google Scholar). The regulation of mRNA is an in the of B. A. PubMed Scopus Google Scholar). The 3′ of are to play a role in regulation of gene expression by mRNA translation and of M. A. L. A. G. A. S. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. Sci. 2004; PubMed Scopus Google Scholar, K. K. K. Abe T. Takeuchi T. J. Immunol. 2003; PubMed Scopus Google Scholar). expression of TCR ζ mRNA with AS 3′ UTR in SLE that it plays an role in the of TCR ζ chain expression in SLE T cells. we to the stability of TCR ζ mRNA with an AS 3′ UTR in SLE T cells and the transport and translation of TCR ζ mRNA with AS 3′ UTR in normal and SLE T cells. In report we that AS 3′ UTR instability to the TCR ζ as it to the mRNA of and to decreased levels of TCR ζ In we that the TCR ζ mRNA with AS 3′ UTR translation to the production of low amounts of protein. the production of TCR ζ mRNA with 3′ UTR a molecular that to decreased expression of TCR ζ chain in SLE T cells. SLE and the of for SLE D.J. N. Arthritis Rheum. PubMed Scopus Google Scholar) for the not to at least the for the SLE patients by the SLE disease patients with from to The of the by the of the involved from and cells from by T cells by of cells a of and to as (17Nambiar M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: PubMed Scopus Google Scholar) In the of T cells in the as by cell The TCR ζ chain of the from The TCR ζ chain J. C. PubMed Scopus Google Scholar) and from from from and from from from of TCR ζ mRNA with and 3′ from T cells from SLE normal to the by the reverse reverse transcription from and as by the The for the by The for the of WT and AS TCR ζ mRNA bp to the of Weissman 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) The 3′ for the of TCR ζ mRNA with WT 3′ UTR bp This is located in the region of the TCR ζ mRNA and is in TCR ζ mRNA with AS 3′ UTR. TCR ζ mRNA with AS 3′ UTR by a that spans of the AS it and not with the WT TCR ζ The sequence of is bp and used as a and the and The reverse transcription product to and used for of TCR ζ mRNA The with a high from in a at for at 1 for and a at for of the and with of the product the of TCR ζ mRNA with and 3′ with a by to the The used to the expression of TCR ζ mRNA with WT and AS 3′ UTR. used for and the in from normal and SLE T and 1 of and for The of of of and in a The for as by with a by to the The for of for by of for for and for for by a that in a the is also separated a to the of The expression for gene by the for the to to a The also amounts of generated for of and the to the gene mRNA in the The generated by and to an from the the of TCR ζ mRNA with WT and AS 3′ UTR as well as from the at and the from the of the in at and TCR mRNA with WT and AS 3′ UTR levels to the and of the and TCR ζ mRNA from T from by and reverse as The TCR ζ mRNA with WT and AS 3′ UTR by bp and bp The a high from in a at for at 1 1 for and a at for The bp WT and bp AS TCR ζ to from and the of the by and AS TCR ζ with to from an the of TCR ζ with WT and AS 3′ UTR to COS-7 COS-7 cells in and at cells and in of of TCR ζ with WT AS 3′ UTR and at in a cells in and cells at with and the mRNA of the cell membrane by (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 and WT and AS TCR ζ mRNA and the as by the of WT AS TCR ζ with the in the of for at The product and to The by with and an and 3′ of WT and AS of TCR ζ mRNA by and the of the luciferase gene in the and from is by an The of the by and of COS-7 and T cells in the The COS-7 cells cells in of with in to to The to and by the and The transfected cells with a luciferase the the transfected cells in a at for and with of by for at with of and of luciferase in a a a in for luciferase by the in by with 1 of a the gene. for the The are as in luciferase compared with that from cells transfected with and analysis of the with the analysis the by the of TCR ζ mRNA with AS 3′ UTR in SLE T and sequence analysis of TCR ζ mRNA the of a TCR ζ chain transcript with alternatively spliced 3′ UTR in human T the TCR ζ mRNA with AS 3′ UTR at low levels in normal human T it to in SLE T cells M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. 2001; PubMed Scopus Google Scholar). Because a 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, M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. Arthritis Rheum. 2001; 44: PubMed Scopus Google Scholar), we the TCR ζ mRNA with AS 3′ UTR to more than the TCR ζ mRNA with WT 3′ UTR. T cells with transcription for of and and the levels of expression of WT and AS 3′ UTR TCR ζ mRNA by as and the of mRNA as a of the at we that the levels of TCR ζ mRNA with AS 3′ UTR compared with the WT ζ mRNA in SLE T cells and also compared the stability of the TCR ζ mRNA with WT 3′ UTR to that with the AS 3′ UTR the of the levels of mRNA to in the stability of AS TCR ζ mRNA is decreased compared with the WT 3′ UTR in SLE T cells of TCR ζ mRNA with AS 3′ UTR in T the the decreased of TCR ζ mRNA with AS 3′ UTR is to SLE T we the stability of TCR ζ mRNA in normal T cells. normal T cells with transcription for of and the levels of TCR ζ mRNA expression with WT and AS 3′ UTR in that the mRNA stability of TCR ζ mRNA with AS 3′ UTR also low in normal T cells and the stability of TCR ζ mRNA with AS 3′ UTR low in normal it not as as it in SLE T cells. of with the of TCR ζ mRNA with AS 3′ UTR and in SLE and normal T and However, the of of TCR ζ mRNA with WT 3′ UTR not significantly between SLE and normal T cells. of TCR ζ mRNA with AS 3′ UTR in SLE and T used a to the stability of TCR ζ mRNA with WT and AS 3′ UTR in SLE T cells and normal T cells with for to the in the stability of the TCR ζ mRNA with WT AS 3′ UTR. The mRNA from the to by reverse The reverse transcription product of TCR ζ and AS 3′ in the of in a as and the the for TCR ζ mRNA with WT and AS 3′ UTR and not The for TCR ζ mRNA with 3′ UTR and mRNA in the in by and the amounts of mRNA from the TCR ζ mRNA with WT AS 3′ UTR as the mRNA in the cells with demonstrated that WT 3′ UTR mRNA more than AS 3′ UTR mRNA the also that TCR ζ mRNA with AS 3′ UTR mRNA in SLE to a compared with WT 3′ UTR mRNA of with in SLE T cells 3′ UTR, and AS 3′ UTR, The stability of TCR ζ mRNA with WT 3′ UTR mRNA and AS 3′ UTR mRNA also in normal T cell by 3′ UTR, and AS 3′ UTR, The that TCR ζ mRNA with WT 3′ UTR to a compared with the TCR ζ mRNA with AS 3′ UTR of with of TCR ζ mRNA with AS 3′ UTR in COS-7 we the stability of TCR ζ mRNA with WT and AS 3′ UTR transfected in COS-7 cells. TCR ζ with WT AS 3′ UTR a expression and transfected to COS-7 cells. of the cells with for and the mRNA by in COS-7 cells of TCR ζ mRNA by by 8 we a in the of TCR ζ mRNA with AS 3′ UTR compared with WT 3′ UTR that the to in from transport of TCR ζ mRNA with WT and AS 3′ UTR from to as a of the transfected cells at with and mRNA of the cell membrane by of TCR ζ mRNA with WT and AS 3′ UTR by The showed that the of TCR ζ mRNA between the and similar at that of TCR ζ 3′ UTR not the transport of the TCR ζ mRNA not of TCR ζ in COS-7 with TCR ζ WT and AS 3′ showed that the of TCR ζ protein in SLE T cells with the of TCR ζ mRNA with AS 3′ UTR M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. 2001; PubMed Scopus Google Scholar). This although the region is in TCR ζ mRNA with AS 3′ UTR, the translational regulation of TCR ζ mRNA is the translational of TCR ζ mRNA with WT and AS 3′ UTR in transfected COS-7 cells by with amounts of the the cells and with a TCR ζ in TCR ζ with WT 3′ UTR a with a molecular of as (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, M.P. Enyedy E.J. Fisher C.U. Krishnan S. Warke V.G. Tsokos G.C. Arthritis Rheum. PubMed Scopus Google Scholar). transfected with TCR ζ AS 3′ UTR produced low amounts of the 16-kDa of TCR ζ protein. analysis showed a in the protein in COS-7 cells transfected with AS TCR ζ However, showed amounts of mRNA in and AS TCR cells. This that of TCR ζ 3′ UTR has a the translation of TCR ζ the also suggest that the region elements that play in the translational regulation of TCR ζ In and of TCR ζ with WT and AS 3′ that the TCR ζ mRNA with AS 3′ UTR in production of protein and the of in the we and the TCR ζ with AS 3′ UTR in vitro as a The in vitro and separated and with to the expression in COS-7 in vitro transcription and translation of TCR ζ with WT 3′ UTR produced a protein of 16-kDa TCR ζ chain The TCR ζ with WT 3′ UTR produced high levels of 16-kDa the TCR ζ with AS 3′ UTR produced significantly low levels of the 16-kDa TCR ζ protein. The also by and the with TCR ζ analysis showed that transfected COS-7 cells with TCR ζ AS 3′ UTR in an in the protein and of with TCR ζ WT and AS 3′ the AS 3′ UTR conferred instability to genes than that of TCR we the WT and AS 3′ of the luciferase gene. TCR ζ WT and AS 3′ UTR by by the with the in and with which is located the luciferase gene at the 3′ region The luciferase by that in of luciferase by sequence analysis not The luciferase TCR ζ WT and AS 3′ UTR with to COS-7 and normal T cells. of with and the luciferase of the transfected cells in and luciferase is significantly decreased in cells transfected with the AS 3′ UTR compared with WT 3′ UTR in and normal T cells and in COS-7 cells by cells and T cells not that AS 3′ UTR instability that is not cell of the of the is the that TCR ζ mRNA with AS 3′ UTR that is in SLE T cells is compared with the WT 3′ UTR in T cells of SLE patients as well as normal and are with the that the the 3′ UTR, the is the K. K. K. Abe T. Takeuchi T. J. Immunol. 2003; PubMed Scopus Google Scholar, G. D.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Res. PubMed Scopus Google Scholar, J. Immunol. 2004; PubMed Scopus Google Scholar). However, compared with normal the of of the TCR ζ mRNA with AS 3′ UTR is more in SLE T cells that the SLE T cells have that TCR ζ mRNA However, the stability of TCR ζ mRNA with WT 3′ UTR is similar between SLE and normal T cells. with the fact that TCR ζ mRNA with AS 3′ UTR is in SLE T cells and TCR ζ chain protein expression with TCR ζ mRNA with AS 3′ UTR, suggest that SLE T cells TCR ζ mRNA with the AS 3′ UTR that is This a for the of TCR ζ mRNA in The levels of AS 3′ UTR TCR ζ mRNA are not increased in T cells from patients with systemic as M.P. Enyedy E.J. Warke V.G. Krishnan S. Dennis G. Kammer G.M. Tsokos G.C. J. 2001; PubMed Scopus Google Scholar). The 3′ UTR of play in gene expression at the M. A. L. A. G. A. S. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. Sci. 2004; PubMed Scopus Google Scholar, N. A. M. A. L. A. A. S. J. PubMed Scopus Google Scholar, Res. Mol. Res. 2004; PubMed Scopus Google Scholar, H. S. J. Biol. 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Res. 2003; PubMed Scopus Google Scholar). is also that in to increased of TCR ζ mRNA with 3′ UTR in SLE T cells. The is that the TCR ζ mRNA with AS 3′ UTR low amounts of 16-kDa TCR ζ protein compared with the ζ chain with WT 3′ UTR and This that the of translational elements in the bp region is required for the and translation of TCR ζ chain more are to the region TCR ζ chain protein are the regulation of protein expression by AS elements S.J. S.J. Morrison J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. S.J. Morrison J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This regulation is by the of protein to the 3′ UTR, and splice deletion of TCR ζ mRNA with AS 3′ UTR the of of the proteins that with the region and have that T cells from SLE patients are in TCR ζ chain and display signaling events (7Liossis S.N. Ding D.Z. Dennis G.J. Tsokos G.C. J. Clin. Investig. 1998; 101: 1448-1457Crossref PubMed Scopus (290) Google Scholar, K. Takeuchi T. N. Pang M. Abe T. J. 1998; PubMed Scopus Google Scholar, T. K. Pang M. Abe T. 1998; PubMed Scopus Google Scholar). The molecular of the TCR ζ chain in SLE T and its in the of the disease has not been findings suggest that expression of TCR ζ mRNA with AS 3′ UTR is with the of TCR ζ chain expression in SLE also an for the regulation of gene expression involved in the by that its In we have that the instability and translation of the AS TCR ζ mRNA to the decreased expression of TCR ζ chain in SLE T cells. that the region of the TCR ζ 3′ UTR elements that proteins that stability and translation and spliced the mRNA and with translational