Alternative splicing of CD45 pre-mRNA is uniquely obedient to conditions in lymphoid cells.

The leucocyte common antigen (LCA or CD45) consists of various isoforms generated by alternative splicing of variable exons 4, 5 and 6 (or A, B and C). To follow splicing behaviour in different cell types we developed a human CD45 mini-gene and analysed its expression in transfected cell lines and transgenic mouse tissues. In Cos-1, HeLa and 3T3 cells we found distinct expression patterns which could only be modulated slightly by protein synthesis inhibitors but not by variation in culture conditions like pH, serum concentration and cell density, or by stimulation with phorbol ester (TPA). In all non-lymphoid transgenic tissues the default splicing pattern (CD45R0) was found, while the expression profile in lymphoid cells, where all eight isoforms are present, mimics that of the endogenous mouse LCA gene products. Next, to examine the factors involved in alternative exon use we analysed the expression pattern of members of the family of SR proteins, well known splicing regulators with arginine/serine-rich (R/S) domains. Cell lines expressed variable levels of SRp75, SRp30 and SRp20 and constant amounts of SRp40. Mouse tissues expressed large amounts of SRp75, SRp55 and SRp40, additional expression of SRp30s and SRp20 was restricted to lymphoid tissues. Therefore, SRp30 and SRp20 may contribute to forming the appropriate cellular conditions for alternative use of CD45 exons 4-6 in the haematopoietic compartment.     

RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.     

Origination of new immunological functions in the costimulatory molecule B7-H3: the role of exon duplication in evolution of the immune system

B7-H3, a recently identified B7 family member, has different isoforms in human and mouse. Mouse B7-H3 gene has only one isoform (2IgB7-H3) with two Ig-like domains, whereas human B7-H3 has two isoforms (2IgB7-H3 and 4IgB7-H3). In this study a systematic genomic survey across various species from teleost fishes to mammals revealed that 4IgB7-H3 isoform also appeared in pigs, guinea pigs, cows, dogs, African elephants, pandas, megabats and higher primate animals, which resulted from tandem exon duplication. Further sequence analysis indicated that this duplication generated a new conserved region in the first IgC domain, which might disable 4IgB7-H3 from releasing soluble form, while 2IgB7-H3 presented both membrane and soluble forms. Through three-dimensional (3D) structure modeling and fusion-protein binding assays, we discovered that the duplicated isoform had a different structure and might bind to another potential receptor on activated T cells. In T cell proliferation assay, human 2IgB7-H3 (h2IgB7-H3) and mouse B7-H3 (mB7-H3) both increased T cell proliferation and IL-2, IFN-γ production, whereas human 4IgB7-H3 (h4IgB7-H3) reduced cytokine production and T cell proliferation compared to control. Furthermore, both h2IgB7-H3 and mB7-H3 upregulated the function of lipopolysacharide (LPS)-activated monocyte in vitro. Taken together, our data implied that during the evolution of vertebrates, B7-H3 exon duplication contributed to the generation of a new 4IgB7-H3 isoform in many mammalian species, which have carried out distinct functions in the immune responses.     

Expression pattern of alternatively spliced PECAM-1 isoforms in hematopoietic cells and platelets

PECAM-1 (CD31) is a cell adhesion molecule that is highly expressed in the endothelium. Hematopoietic cells including platelets, monocytes, neutrophils, and some T cells also express moderate levels of PECAM-1. PECAM-1 undergoes alternative splicing generating a number of isoforms in the endothelium. However, the expression of PECAM-1 isoforms in hematopoietic cells and platelets has not been determined. Here, we examined the expression pattern of PECAM-1 isoforms in human and rodent hematopoietic cells and platelets by RT-PCR and DNA sequencing analysis. Our results showed that multiple PECAM-1 isoforms are expressed in a cell-type and species-specific pattern. We identified seven human PECAM-1 isoforms, six murine PECAM-1 isoforms, and four rat PECAM-1 isoforms. The full-length PECAM-1 was the predominant isoform detected in human cells. The PECAM-1 isoforms that lack exon 14 and 15 (delta14&15) or delta12,14&15 were the predominant isoform in rodent cells. In addition, we identified a novel PECAM-1 isoform, delta13&14, in human hematopoietic cells. Thus, hematopoietic cells express multiple isoforms of PECAM-1 in a pattern similar to that observed in the endothelium of the same species. The regulated expression of these isoforms may be important during hematopoiesis and transendothelial migration.     

Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule that is highly expressed on the surface of endothelial cells and some hematopoietic cells. Its cytoplasmic domain is encoded by multiple exons, which undergo alternative splicing. Here, we demonstrate that the human PECAM-1 cytoplasmic domain undergoes alternative splicing, generating six different isoforms. RT-PCR cloning and DNA sequence analysis indicated that human tissue and endothelial cells express multiple isoforms of PECAM-1, including the full-length PECAM-1 and five other isoforms, which lack exon 12, 13, 14, or 15 or exons 14 and 15. The full-length PECAM-1 is the predominant isoform detected in human tissue and endothelial cells. This is in contrast to murine endothelium, in which the PECAM-1 isoform lacking exons 14 and 15 is the predominant isoform. The PECAM-1 isoform lacking exon 13 detected in human tissue and endothelial cells is absent in murine endothelium. The expression pattern of PECAM-1 isoforms changes during tube formation of endothelial cells on Matrigel, which may indicate specialized roles for specific isoforms of PECAM-1 during angiogenesis. The data presented here demonstrate that human PECAM-1 undergoes alternative splicing, generating multiple isoforms in vascular beds of various tissues. Therefore, the regulated expression of these isoforms may influence endothelial cell adhesive properties during angiogenesis and/or vasculogenesis.     

Carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes

Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), the primordial carcinoembryonic Ag gene family member, is a transmembrane cell adhesion molecule expressed in leukocytes, epithelia, and blood vessel endothelia in humans and rodents. As a result of differential splicing, CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, that have long (L) or short (S) cytoplasmic domains, respectively. The L:S expression ratios vary in different cells and tissues. In addition to CEACAM1, human but not rodent cells express GPI-linked CEACAM members (CEACAM5-CEACAM8). We compared the expression patterns of CEACAM1-L, CEACAM1-S, CEACAM6, and CEACAM8 in purified populations of neutrophilic granulocytes, B lymphocytes, and T lymphocytes from rats, mice, and humans. Human granulocytes expressed CEACAM1, CEACAM6, and CEACAM8, whereas human B lymphocytes and T lymphocytes expressed only CEACAM1 and CEACAM6. Whereas granulocytes, B cells, and T cells from mice and rats expressed both CEACAM1-L and CEACAM1-S in ratios of 2.2-2.9:1, CEACAM1-S expression was totally lacking in human granulocytes, B cells, and T cells. Human leukocytes only expressed the L isoforms of CEACAM1. This suggests that the GPI-linked CEACAM members have functionally replaced CEACAM1-S in human leukocytes. Support for the replacement hypothesis was obtained from experiments in which the extracellular signal-regulated kinases (Erk)1/2 were activated by anti-CEACAM Abs. Thus, Abs against CEACAM1 activated Erk1/2 in rat granulocytes, but not in human granulocytes. Erk1/2 in human granulocytes could, however, be activated by Abs against CEACAM8. We demonstrated that CEACAM1 and CEACAM8 are physically associated in human granulocytes. The CEACAM1/CEACAM8 complex in human cells might accordingly play a similar role as CEACAM1-L/CEACAM1-S dimers known to occur in rat cells.     

Uniquely conserved non-translated regions are involved in generation of the two major transcripts of protein phosphatase 2Cbeta

Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.