Abstract
CD45, a receptor-type protein tyrosine phosphatase involved in lymphocyte activation, consists of five isoforms generated by alternative mRNA splicing. The pattern of CD45 splicing depends upon cell type, state of activation, and thymic development. We previously showed that mouse B and thymocyte cell lines transfected with a human CD45 minigene construct appropriately splice the alternative exons, demonstrating the presence of trans-acting tissue-specific splicing factors. To study the regulation of CD45 alternative splicing, cells having different splicing patterns were transiently fused, and mRNA was analyzed using reverse transcription-polymerase chain reaction. Human B cells, normally producing only the largest CD45 mRNAs, could splice out the alternative exons after fusion with mouse thymocytes. In contrast, the splicing pattern of human T cell lines (smaller CD45 isoforms) was unaltered by fusion with mouse B cells. This suggests that cells expressing the smallest CD45 isoform contain negatively acting trans-factor(s) that allow the alternative exons to be skipped, and that the full length isoform containing all three alternative exons is the default pattern of splicing. In agreement, incubation of thymocytes or T cell subsets with cycloheximide increased mRNA corresponding to the larger isoforms. CD45 isoform expression can now be interpreted in terms of the presence or absence of negative regulatory trans-acting splicing factors.
Highlights
The Alternative Splicing of the CD45 Tyrosine Phosphatase Is Controlled by Negative Regulatory trans-Acting Splicing Factors*
Of activation, and thymic development.We previously Some anti-CD45 mAbs recognize epitopes common to all showed that mouse B and thymocyte cell lines transfected with a human CD46 minigene construct appropriatelysplicethe alternative exons, demonstrating thepresenceof trans-acting tissue-specific splicing factors
To study the regulation of CD46 alternative splicing, cells having different splicing patterns were transiently fused, and mRNA was analyzed using reverse transcription-polymerase chain reactioHnu.man of the isoforms, while others, designated antiCD45RA, recognize exon A which is present in the two highest M, isoforms containing alternativeexons 4 and 5 or exons 4, 5, and 6
Summary
Cell Lines and Media-The human leukemic T cell lines, Jurkat, HPB-ALL, and HSB2, and B cell lines, Raji and Daudi, were used. 300-19cells transfected with the pZipneo plasmid vector (300-19-pZip)or transfected with cDNA for each of the individual human CD45 isoforms were previously described [39]. They include: 300-19-LCA.l, expressing the 180-kDa isoform having no alternative exons (0-exon); 300-19-LCA.623,expressing the 190-kDa alternative exon 5-containing isoform; 300-19-LCA.260, expressing the 205-kDa exon 66-containing isoform; 300-19-LCA.338,expressing the 205-kDa exon 46 isoform; and 300-19-LCA., expressing the 220-kDa exon 456 isoform. 300-19cells were transfected with the pSV2neo-SP plasmid vector (300-19-pSVneo)or were co-transfected with pSV2neo-SP and the human LCA minigene construct, pSV-mini-LCA., which contains human CD45 exons in the following sequence: 236789 and is designated 300-19-LCA. EL4 cells were transfected with pSV2neo-SP plasmid vector alone (EL4-pSVneo) or were co-transfected with pSV2neo-SP and the human LCA minigene construct, pSV-mini-. The production of these vectors, minigene constructs, and transfectants is described in Ref. 29
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