Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation

Abstract

We recently isolated the gene and a cDNA clone for the mouse T-cell surface antigen Lyt-2 and showed that Lyt-2 is homologous to the human Leu-2 (T8) antigen and that the gene encoding it is a member of the immunoglobulin gene superfamily. By screening a mouse thymus cDNA library with the Lyt-2 cDNA clone, we isolated two classes of cDNA clones, alpha and alpha', which differ by 31 base pairs. Comparison of the alpha cDNA with genomic sequence data indicates that there are five exons encoding Lyt-2: a fused leader/immunoglobulin variable region-like exon, a spacer region exon, a transmembrane exon, and two cytoplasmic exons. The alpha' cDNA clones lack the first of the two cytoplasmic exons and have a direct splice from the donor splice site of the transmembrane exon to the acceptor of the second cytoplasmic exon. This splice changes the reading frame for the second cytoplasmic exon, causing a stop codon shortly after the splice so that the alpha' cDNA clone codes for a peptide 25 residues shorter than the alpha cDNA-encoded peptide. We have constructed expression vectors with alpha and alpha' cDNAs and have shown that L-cell transfectants of these produce Lyt-2 polypeptides of the predicted sizes and that these associate as homodimers on the cell membranes. We found the two species of mRNA corresponding to alpha and alpha' cDNAs at equal levels in thymus RNA by using S1 nuclease analysis. Although lymph node T cells have only the alpha form of Lyt-2 protein, S1 nuclease analysis shows that lymph nodes have about 20% alpha' mRNA relative to alpha. Thus, Lyt-2 is regulated at RNA processing, translational, and/or post-translational steps.