Zilch, Christian Frank; (1998) The control of alternative splicing of the leucocyte common antigen (CD45). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The leucocyte common antigen (CD45) is a high molecular weight transmembrane glycoprotein exclusively expressed on hematopoietic cells. It plays an important role in the signal transduction of T cells, in which various alternatively spliced isoforms are generated according to the stage of development and activation. Monoclonal antibodies recognising high and low molecular weight isoforms have been used to distinguish between naive and memory T cells. The splicing pattern of CD45 in T cells is altered in some individuals, who show continuous expression of high molecular weight isoforms containing exon A on activated T cells. A silent point mutation at position 77 of CD45 exon A was shown to be associated with the variant splicing pattern. This thesis provides strong evidence that this mutation is the cause of abnormal splicing. In order to study the factors influencing CD45 alternative splicing, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced. Clones expressing different isoforms of CD45 were isolated in which expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. In addition, minigenes including this mutation were constructed and transfected into various cell lines. Semi-quantitative RT-PCR showed a striking increase in splicing to CD45RA when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. Another important question is how the alternatively spliced exons of CD45 are controlled. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA from minigenes was investigated by cotransfection assays of several cloned splicing factors belonging to the family of SR proteins. It was clearly shown that SRp 20 and SRp30c have antagonistic effects on CD45 alternative splicing. SRp 20 induced shifts in the CD45RA form, whereas SRp30c promoted splicing in the opposite direction in a dose dependent manner resulting in CD45RO expression. SR proteins consist of a modular structure with single or multiple RNA recognition motifs (RRMs) and one serine-arginine rich domain. Experiments using domain swap chimera between SRp30c and SRp30a as well as deletion mutants of SRp30c indicate that the specificity for CD45 exon A splicing is contained within the second RRM of SRp30c. Collectively, these findings imply that individual members of the SR family of splicing regulators can specifically inhibit or promote the splicing of alternative CD45 exons. A general mechanistic model for alternative splice site selection has been proposed.

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