Dissecting alternative splicing in the formation of Miltenberger glycophorin subtype III (GYP.Mur).

Abstract

Miltenberger subtype III (Mi.III, GP.Mur) is one of the most important red cell phenotypes in the fields of transfusion in South-East Asia. GP.Mur is believed to evolve from homologous gene recombination events between glycophorin A (GYPA) and glycophorin B (GYPB). GYP.Mur differs from GYPB in only seven nucleotides dispersed near the region of 3' exon 3 of GYP.Mur. The goal of this study was to dissect how these nucleotide variants affected splicing of exon 3. We first designed two minigene constructs: one containing GYP.Mur from exon 2 to exon 4 and the other containing GYPB in the same region. To test how these nucleotide variations between GYP.Mur and GYPB affected the splicing, a repertoire of the GYP.Mur-like minigene constructs with different point mutations were created. These minigene variants were evaluated for their abilities to induce splicing of exon 3 using a heterologous expression system. (1) GYP.Mur minigene expressed exons 2, 3 and 4, whereas GYPB minigene expressed only exon 2 and exon 4. (2) The single nucleotide alteration at the position of the 5' splice site of glycophorin intron 3 reversed the splicing decision. (3) The nucleotide variations between GYP.Mur and GYPB other than that at the 5' splice site showed very little or no effect on splicing of exon 3. Splicing of the glycophorin B-A-B hybrids (GYP.Mur and GYP.BUN) and unsplicing of GYPB follow the GU-AG rule strictly.