Background/Purpose T cells from patients with systemic lupus erythematosus (SLE) express reduced amounts of the critical CD3 zeta signaling chain, and are poor producers of the vital cytokine interleukin (IL)-2.  Using oligonucleotide-pulldown and mass spectrometry approaches, we previously identified the serine arginine-rich splicing factor 1 (SRSF1) binding to the 3`UTR of CD3 zeta mRNA. We showed that SRSF1 regulates alternative splicing of the CD3 zeta 3`UTR to promote a full length 3`UTR over an unstable splice variant, and thus promotes expression of CD3 zeta chain. SLE T cells exhibit reduced expression of SRSF1, more so patients with worse disease as evidenced by higher SLE disease activity index (SLEDAI). SRSF1 and the serine arginine (SR) proteins have recently been suggested to regulate transcription, in addition to regulating post-transcriptional events in gene expression such as alternative splicing, mRNA stability and translation. CD3 zeta is regulated by the Ets transcription factor family member E-74-like factor (Elf)-1 via two Ets binding sites (EBS) within the CD3 zeta promoter. In this study, we asked whether SRSF1 could activate CD3 zeta transcription and if this involved the transcription factor Elf-1.

Methods T cells were isolated by negative selection from peripheral blood of healthy individuals, and patients with SLE. Primary T cells were transfected with an SRSF1 or empty expression plasmid by nucleofection. mRNA and protein expression were assessed by real time quantitative pcr and immunoblotting respectively. To measure transcriptional activity, CD3 zeta promoter-luciferase constructs were co-transfected with increasing amounts of SRSF1 into 293T cells or into primary T cells, and luciferase activity measured using the dual luciferase assay system. Reporter chromatin immunoprecipitation (R-ChIP) assays were used to assess recruitment of proteins to the CD3 zeta promoter-luciferase construct. Specific antibodies were used to immunoprecipitate SRSF1, HA (tag present in the SRSF1 plasmid), and Elf-1 proteins. Quantitative real time pcr was performed to assess enrichment, and normalized to values obtained from inputs.

Results We show that SRSF1 expression directly correlates with CD3 zeta chain expression in T cells from patients with SLE. Overexpression and silencing of SRSF1 in primary T cells results in corresponding increase and decrease in total CD3 zeta mRNA expression. SRSF1 increases activity of the CD3 zeta promoter-luciferase in a dose dependent manner. SRSF1 overexpression leads to increased Elf-1 expression in T cells. Recruitment of SRSF1 and Elf-1 (a known transcriptional activator of CD3 zeta) to the CD3 zeta promoter is observed in T cells that overexpress SRSF1 compared to control transfected cells. 

Conclusion Our results reveal a novel transcriptional mechanism by which SRSF1 regulates CD3 zeta and may represent a molecular mechanism that contributes to the reduced CD3 zeta expression in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/serine-arginine-rich-splicing-factor-1-srsf1-regulates-transcriptional-activation-of-the-t-cell-receptor-cd3-zeta-chain-in-human-t-cells/