Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease that is characterized by the Philadelphia chromosome (Ph), which is generated by the reciprocal translocation t(9;22)(q34;q11) that results in the fusion of the c-abl oncogene 1 (ABL1) with the breakpoint cluster region (BCR) gene. T cell immunodeficiency including thymic output function, abnormal T cell receptor (TCR) repertoire expression and, in part, abnormal TCR signal transduction, such as that involving the TCRζ chain is found in patients with CML[2–6], and T cell function becomes suppressed as the disease progresses in some patients.
The TCR/CD3 complex plays a central role in T cell activation. This complex comprises of two chains, αβ or γδ, these chains are noncovalently associated with CD3 subunits, which include four transmembrane proteins: CD3γ, CD3δ, CD3ε and CD3ζ (also referred to as TCRζ). These subunits are known to form three distinct dimers, CD3γε, CD3δε, and CD3ζζ, to mediate TCR signal transduction[7–10]. There are four tyrosine kinase families involved in TCR signal transduction including the Csk, Src, Tec, and ZAP-70 (CD3 zeta chain associated protein kinase 70 kDa) kinase families. ZAP-70 is a cytosolic protein that is recruited to the T cell plasma membrane following TCR stimulation and binds to phosphorylated TCRζ immunoreceptor tyrosine-based activation motifs (ITAMs); it plays a critical role in activating downstream T cell signal transduction pathways following TCR engagement. There is evidence that the Fce receptor type Iγ (FcεRIγ) chain, which is a member of the TCRζ chain protein family and a component of the high-affinity IgE receptor, can replace a functionally deficient TCRζ chain and facilitate TCR/CD3 complex-mediated signaling[12, 13].
The absence of the TCRζ chain not only influences the TCR expression on the cell membrane and the number of single positive (i.e., CD4+ or CD8+) circulating T cells, it also impairs the proliferative response and the mature T cell activation level. T cells from patients with leukemia are functionally impaired, and this is related to decrease TCRζ chain expression[2, 3, 5, 14]. Recently, we has reported the expression pattern of the four CD3 genes in patients with AML and CML[2, 4, 5, 15], and it has been reported that the aberrant TCRζ chain expression found in the T cells of patients with systemic lupus erythematosus (SLE) may be associated with the decreased stability and translation of a TCRζ mRNA with an alternatively spliced 3'-untranslated region. However, the mechanism of TCRζ deficiency in T cells in patients with cancer remains unclear.
The TCRζ gene spans 31 kb in the chromosome 1q23.1 locus and has eight exons that are separated by introns ranging from 700 bp to greater than 8 kb[17, 18]. The TCRζ mRNA is a 1,472 kb spliced product of the eight exons with a 492 bp coding region and a long downstream 906 bp 3'-untranslated region (UTR), which is encoded by exon VIII. The stability of the TCRζ mRNA is mainly influenced by the downstream 3’ untranslated region and poly A tail. The 906 bp TCRζ 3'-UTR has several polyadenylation sites. Exon VIII comprises 20 amino acids of the carboxy-terminus and the 3'-UTR of TCRζ chain. Recently, it has been reported that there are several TCRζ chain isoforms with different 3'-UTR nucleotide sequences in mouse T cells. The activation of alternative splicing within the 3'-UTR through two internal (5' and 3') splice sites results in a splice deletion of 562 bases (nucleotides 672–1233), leading to the generation of a 344 bp alternatively spliced (AS) variant. The 344 bp AS TCRζ isoform lacks two critical regulatory adenosine/uridine-rich elements (ARE) and a translation regulatory sequence. The stability and translation of this isoform are significantly lower than that of the 906 bp WT TCRζ isoform; consequently, the relative amount of TCRζ protein generated by the AS isoform is significantly lower than that from the WT isoform[22, 23].
T cells from healthy individuals predominantly express the wild type (WT) isoform, whereas an increased level of the AS isoform was reported in T cells from SLE patients. Frequent mutations/polymorphisms and aberrant splicing of the downstream 3'-UTR may affect the stability and/or transport of the TCRζ chain mRNA, leading to its downregulation in SLE T cells. Although differential expression of the TCRζ 3'-UTR isoforms contributes to differential TCRζ protein expression levels, the factor(s) regulating the alternative splicing of the TCRζ 3'-UTR is unknown.
Alternative splicing is a powerful gene regulation mechanism that results in the generation of numerous transcripts and proteins from a single gene. Splice site selection is regulated by cis-acting elements such as intronic and exonic splicing enhancer and silencer sequences, respectively[25, 26]. Alternative splicing factor/splicing factor 2 (ASF/SF2) is a prototypical SR protein that was originally identified in HeLa nuclear extract as a factor required to reconstitute splicing in S100 cellular extract[27, 28] and influence alternative splicing site selection in a concentration-dependent manner[28, 29]. ASF/SF2 acts early during spliceosome assembly and participates in multiple steps during constitutive splicing and the regulation of alternative splicing by interacting with the pre-mRNAs and/or other splicing factors[28, 30, 31]. ASF/SF2 regulates the alternative splicing of eukaryotic genes such as caspase-9 and the T cell differentiation marker CD45[19, 32, 33]. Recently, the involvement of ASF/SF2 in the post-transcriptional regulation of TCRζ was described in T cells from patients with SLE. ASF/SF2 binds to the 3'-UTR of TCRζ and regulates the shift in alternative splicing from the AS to the WT isoform and regulates TCRζ protein expression.
Based on our previous finding that the TCRζ chain gene expression level was significantly decreased in CML, we further investigated the expression pattern of the TCRζ regulating factors TCRζ 3'-UTR and ASF/SF-2, as well as the expression level and correlation of FcεRIγ and ZAP-70, to evaluate the ASF/SF-2 regulating effects of TCRζ 3'-UTR formation.