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

Bhabadeb Chowdhury,Christos G Tsokos,Sandeep Krishnan,James Robertson,Carolyn U Fisher,Rahul G Warke,Vishal G Warke,Madhusoodana P Nambiar,George C Tsokos

doi:10.1074/jbc.m501048200

Abstract

The molecular mechanisms involved in the aberrant expression of T cell receptor (TCR) zeta chain of patients with systemic lupus erythematosus are not known. Previously we demonstrated that although normal T cells express high levels of TCR zeta mRNA with wild-type (WT) 3' untranslated region (3' UTR), systemic lupus erythematosus T cells display significantly high levels of TCR zeta mRNA with the alternatively spliced (AS) 3' UTR form, which is derived by splice deletion of nucleotides 672-1233 of the TCR zeta transcript. Here we report that the stability of TCR zeta mRNA with an AS 3' UTR is low compared with TCR zeta 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 zeta with AS 3' UTR produced low amounts of 16-kDa protein. In vitro transcription and translation also produced low amounts of protein from TCR zeta with AS 3' UTR. Taken together our findings suggest that nucleotides 672-1233 bp of TCR zeta 3' UTR play a critical role in its stability and also have elements required for the translational regulation of TCR zeta chain expression in human T cells.

Highlights

From the ‡Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500 and the ¶Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
The T cell receptor (TCR) ␨ mRNA with alternatively spliced (AS) 3Ј untranslated region (UTR) was detected at low levels in normal human T cells, it was found to be predominantly expressed in systemic lupus erythematosus (SLE) T cells [18]
Purified T cells were incubated with transcription inhibitor actinomycin D (5 ␮g/ml) for different periods of time (0, 1, 2, and 4 h), and the levels of expression of WT and AS 3Ј UTR TCR ␨ mRNA were quantitated by semiquantitative RT-PCR using specific primers as described under “Materials and Methods.”

Summary

Introduction

Creased free intracytoplasmic calcium levels [11] and tyrosine phosphorylation of cytosolic proteins [7] This “overexcitability” of the SLE T cell has been attributed to the fact that the missing TCR ␨ chain is replaced by the FcR␥ chain [12] and the surface membrane lipid rafts are aggregated [13]. Nucleotide sequence analysis of the TCR ␨ gene showed increased frequency of alternatively spliced (AS) forms missing various exons as well as splice insertion in SLE T cells [17]. Using a specific primer that spans either side of the newly identified alternatively spliced site, we recently reported that the AS form of TCR ␨ mRNA with 344 bp 3Ј UTR was predominantly expressed in SLE T cells compared with normal T cells [14]

Methods

Results

Conclusion