Serine Arginine-Rich Splicing Factor 1 (SRSF1) Contributes to the Transcriptional Activation of CD3ζ in Human T Cells

Abstract

T lymphocytes from many patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor (TCR)-associated CD3 zeta (ζ) signaling chain, a feature directly linked to their abnormal phenotype and function. Reduced mRNA expression partly due to defective alternative splicing, contributes to the reduced expression of CD3ζ chain. We previously identified by oligonucleotide pulldown and mass spectrometry approaches, the serine arginine-rich splicing factor 1 (SRSF1) binding to the 3' untranslated region (UTR) of CD3ζ mRNA. We showed that SRSF1 regulates alternative splicing of the 3'UTR of CD3ζ to promote expression of the normal full length 3`UTR over an unstable splice variant in human T cells. In this study we show that SRSF1 regulates transcriptional activation of CD3ζ. Specifically, overexpression and silencing of SRSF1 respectively increases and decreases CD3ζ total mRNA and protein expression in Jurkat and primary T cells. Using promoter-luciferase assays, we show that SRSF1 enhances transcriptional activity of the CD3ζ promoter in a dose dependent manner. Chromatin immunoprecipitation assays show that SRSF1 is recruited to the CD3ζ promoter. These results indicate that SRSF1 contributes to transcriptional activation of CD3ζ. Thus our study identifies a novel mechanism whereby SRSF1 regulates CD3ζ expression in human T cells and may contribute to the T cell defect in SLE.

Fig 1. Correlation between SRSF1 and CD3ζ chain expression in T cells from patients with SLE.

(A) Peripheral blood T cells from SLE patients (“L”, n = 29) and healthy individuals (“N”, n = 23) were lysed and total protein immunoblotted for SRSF1, CD3ζ and β-actin. Representative blots are shown. (B) Densitometric quantitation of SRSF1 and CD3ζ blots were performed and normalized to β-actin. SRSF1 and CD3ζ expression of SLE patients were normalized to those from healthy individuals and the relative values were plotted on an x/y graph.

Fig 2. SRSF1 regulates CD3ζ protein and mRNA expression.

(A) Jurkat cells were transfected with increasing concentrations of SRSF1 specific siRNA (siSRSF1) or non-silencing control siRNA (siCtrl). 24–48 hrs post transfection, cells were lysed and total protein immunoblotted for SRSF1, CD3ζ and β-actin. (B) Densitometric quantitation of SRSF1 and CD3ζ blots was performed and normalized to β-actin. Graphs show average values from three independent experiments and error bars represent SEM. (C) Primary T cells were transfected with siSRSF1 or siCtrl siRNA, and cells were collected 24hrs post transfection. Total RNA was reverse transcribed and quantitative real time pcr performed using specific primers for CD3ζ and Cyclophilin A. Graphs show average values from five independent experiments and error bars represent SEM. (D) Primary T cells were transfected with SRSF1 expression plasmid (pSRSF1) or an empty plasmid as control (pcDNA). Cells were collected 24hrs post transfection. Total RNA was reverse transcribed and quantitative real time pcr performed using specific primers for CD3ζ and Cyclophilin A. Graphs show average values from nine independent experiments and error bars represent SEM. Asterisks indicate p <0.05.

Fig 3. SRSF1 regulates transcriptional activity of the CD3ζ promoter in 293T cells.

(A) Schematic showing luciferase constructs with different lengths of the CD3ζ promoter. (B) 293T cells were co-transfected with an empty pGL2-basic plasmid or the indicated CD3ζ promoter-luciferase constructs and empty vector (pcDNA) or SRSF1 expression vector (pSRSF1) in increasing concentrations such that the ratio of the pcDNA or pSRSF1 (i.e., effector) plasmid to the luciferase (i.e., reporter) plasmid was 2:1 or 3:1. 24hours post transfection, cells were lysed, and luciferase activity measured using the dual-luciferase assay. Graphs show average values from at least three independent experiments and error bars represent SEM. Asterisks indicate p <0.05; NS = not significant.

Fig 4. SRSF1 regulates transcriptional activity of the CD3ζ promoter in primary human T cells.

(A) Schematic showing the CD3ζ promoter-luciferase (pGL2-Zeta-3) construct. (B) Peripheral blood T cells were transfected with the pGL2-Zeta-3 reporter construct and co-transfected with either empty vector (pcDNA) or an SRSF1 expression vector (pSRSF1) in increasing concentrations such that the ratio of the pcDNA or pSRSF1 (i.e., effector) plasmid to the luciferase (i.e., reporter) plasmid was 3:1, 4:1, or 5:1. 4 hours post transfection, cells were lysed and luciferase activity measured using the dual-luciferase assay. Graphs show average values from three independent experiments and error bars represent SEM. Asterisks indicate p <0.05.

Fig 5. SRSF1 is recruited to the CD3ζ promoter.

(A) Schematic showing the CD3ζ promoter-luciferase pGL2-Zeta-3 construct. Arrows indicate primers used for ChIP pcr amplification. (B) 293T cells were co-transfected with the CD3ζ promoter-luciferase construct and empty vector (pcDNA) or SRSF1 expression vector (pSRSF1). At 24hrs post transfection, cells were collected and ChIP assays performed using an SRSF1- specific antibody, HA-tag specific antibody or a control IgG antibody. The CD3ζ promoter-luciferase region was amplified by quantitative real time pcr and normalized to the input DNA. Graphs show average values from at least three independent experiments and error bars represent SEM. (C) Primary T cells were co-transfected and ChIP assays performed as in A, using an HA-tag specific antibody or a control IgG antibody. The CD3ζ promoter-luciferase region was amplified by quantitative real time pcr and normalized to the input DNA. Graphs show average values from at least four independent experiments and error bars represent SEM. Asterisks indicate p <0.05.