ACTIVATION OF A CRYPTIC SPLICE-SITE IN INTRON-24 LEADS TO THE FORMATION OF APOLIPOPROTEIN B-27.6

Apo B expression is confined to the intestine and liver, and its secre tion from these tissues is dependent on the expression of a lipid tran sfer protein, microsomal triglyceride transfer protein (MTP). Previous ly, we reported a model system for the study of apolipoprotein (ape B) biogenesis using heterologous expression in COS cells (Patel SB, Grun dy SM. J. Lipid Res. 1995;36:2090-2103). We now report the characteriz ation of the effects of a T-->C transition in the splice-site at + 2 o f intron 24 previously reported by Talmud et al. (J. Lipid Res. 1994;3 5:468-77). Using our heterologous expression system, we show that the mutation led to aberrant processing of intron 24, but normal processin g of intron 25. The resultant translation of this mutant mRNA produced a truncated apo B protein of the size of apo B-27.6. Reverse transcri ption, polymerase chain reaction and sequencing of the amplified produ cts were used to show that a cryptic donor splice-site within intron 2 4 was utilized, resulting in the generation of a novel hydrophilic 29 amino acid carboxyl-terminal tail. Co-expression of apo B-27.6 with mi crosomal triglyceride transfer protein (MTP) showed that this protein could bind MTP and resulted in the secretion of a lipoprotein particle with a buoyant density in the range 1.16-1.25 g/ml. These results ind icate that this splice-site mutation leads to an activation of a downs tream cryptic splice-site within intron 24, causing an insertion of 40 bases of intron 24 sequences into the mature RNA. This leads to a fra me-shift of translation resulting in addition of 29 new amino acids at the carboxyl-terminus, before an in-frame stop translation codon is e ncountered, truncating the apo B at B-27.6. (C) 1997 Elsevier Science Ireland Ltd.