New CD3ζ allele showing a 9-bp deletion in the 3 ′ -UT region of the gene A. Aguinaga-Barrilero 1 , N. Rodr´ ıguez-P´ erez 1 , M. P ´ erez-Blas 1 , A. Guti ´ errez-Calvo 2 & J. M. Mart´ ın-Villa 1 1 Inmunolog´ ıa, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain 2 Servicio de Cirug´ ıa General y Aparato Digestivo, Hospital Universitario Pr´ ıncipe de Asturias, Alcal´ a de Henares, Madrid, Spain Key words: AU-rich elements; CD3ζ; deletion; messenger RNA stability We report here a non-previously described 9-bp deletion in the 3 ′ -UT region of the CD3ζ gene, located in between two AREs. The CD3ζ chain of the T-cell receptor (TCR)–CD3 com- plex is a 16 kDa transmembrane protein expressed by nat- ural killer (NK), natural killer T (NKT) and T-cells, with a short extracellular domain, a transmembrane region and a long cytoplasmic tail, which contains three immunorecep- tor tyrosine-based activation motifs and is involved in signal transduction upon TCR ligation and subsequent activation of T lymphocytes (1). T lymphocytes are cells of the adaptive immune response that play a crucial role in the defense against pathogens and tumoral antigens. The expression of this chain on T-cells is downregulated in several clinical conditions such as sys- temic lupus erythematosus [SLE (2)], cancer (3) or chronic inflammation (4). Stability of the corresponding messenger RNA (mRNA) is one of the mechanisms proposed to explain this decreased CD3ζ expression. Adenosine- and uridine-rich elements (AUUUA motifs, AREs) located in 3 ′ -untranslated (3 ′ -UT) regions are the best determinants of mRNA insta- bility (5). These elements have also been shown to control mRNA translation and, hence, protein concentration. This is achieved by means of AREs-binding proteins (AREs-BP) that regulate the fate of the mRNA, either destabilizing it (as is the case with ARE/poly(U)-binding/degradation fac- tor 1, AUF-1) or stabilizing it (Hu-antigen R, Hu-R pro- tein). Thus, changes in the AREs sequence may affect the ability of AREs-BP to bind to its target and hence mRNA stability. In this regard, mice with natural polymorphisms in the tumor necrosis factor (TNF)-α 3 ′ -UT region, includ- ing the ARE, exhibit lower levels of TNF-α production because of altered mRNA translation (5). Recently, and more relevant to the present manuscript, an alternatively spliced form of the CD3ζ mRNA with a 562-base long (672–1233 nucleotide) deletion has been described in SLE patients. This alternatively spliced form lacks two of the three AREs of the CD3ζ mRNA, and Hu-R is then unable to bind to it. As a consequence, the mRNA is significantly more unsta- ble and has a lower protein expression than the wild-type mRNA (6). We report here a new mRNA sequence with a 9-bp deletion close to two ARE in the 3 ′ -UT region of the CD3ζ mRNA. Blood samples were obtained by venopunction from an 82- year-old patient with cutaneous angiosarcoma and gastric metastases, who died shortly after blood sample was drawn. Peripheral blood mononuclear cells (PBMC) were isolated in Ficoll-Paque gradient. DNA and RNA were isolated from PBMC using Trizol reagent following manufacturer’s indi- cations. RNA was retrotranscribed to complementary DNA (cDNA) using first strand cDNA synthesis kit (Roche, Indi- anapolis, IN). Genomic and cDNA were PCR amplified using CD3ζ-specific primers and sequenced at the Universidad Complutense de Madrid (UCM) DNA Sequencing Facility (CAI Gen´ omica). cDNA sequencing disclosed the presence of two different CD3ζ mRNA molecules, one corresponding to the consen- sus sequence and the other one showing a 9-bp deletion in the 3 ′ -UT region, in between the second and third AREs. To assess whether this RNA was an alternatively spliced form or transcription of a corresponding gene, genomic DNA was sequenced. The patient turned out to be heterozygous with regard to the CD3ζ gene presenting a wild-type gene and a gene with a 9-bp deletion affecting exon 8 of the gene, span- ning from nucleotide 89148 to 89156 (Figure 1). This new sequence was submitted to the GenBank, and the accession number EF079882 assigned. Given the importance of AREs in mRNA stability, and thus in protein expression, mfold software (accessible at http://mfold.bioinfo.rpi.edu/) was used to analyze the effect this 9-bp deletion could exert at the mRNA level. Results predicted that according to the free energy value (G, a measure of molecular stability), this mRNA would have a clear altered secondary structure and lower stability (G =−581.6) when compared with the non-deleted CD3ζ mRNA (G =−592.06), a feature that could conceivably affect protein expression level. In the patient, the expression of the CD3 complex (but not the CD3ζ chain) was assessed by flow cytometry using a monoclonal antibody specific for the CD3ε chain, yielding a normal value. Although an altered CD3ζ expression cannot be ruled out, the patient would most probably possess normal levels of this protein because the mutation is present in heterozygosis. Transfection experiments into CD3ζ-defective Jurkat cells using this CD3ζ 9bp-deleted cDNA will help clarify this issue. 356  2009 John Wiley & Sons A/S · Tissue Antigens 74, 343–357