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 secretion from these tissues is dependent on the expression of a lipid transfer protein, microsomal triglyceride transfer protein (MTP). Previously, we reported a model system for the study of apolipoprotein (apo B) biogenesis using heterologous expression in COS cells (Patel SB, Grundy SM. J. Lipid Res. 1995;36:2090-2103). We now report the characterization of the effects of a T-->C transition in the splice-site at +2 of intron 24 previously reported by Talmud et al. (J. Lipid Res. 1994;35:468-77). Using our heterologous expression system, we show that the mutation led to aberrant processing of intron 24, but normal processing 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 transcription, polymerase chain reaction and sequencing of the amplified products were used to show that a cryptic donor splice-site within intron 24 was utilized, resulting in the generation of a novel hydrophilic 29 amino acid carboxyl-terminal tail. Co-expression of apo B-27.6 with microsomal 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 indicate that this splice-site mutation leads to an activation of a downstream cryptic splice-site within intron 24, causing an insertion of 40 bases of intron 24 sequences into the mature RNA. This leads to a frame-shift of translation resulting in addition of 29 new amino acids at the carboxyl-terminus, before an in-frame stop translation codon is encountered, truncating the apo B at B-27.6.
Nemeth-Slany
A
A
Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX 75235-9052, USA.
Talmud
P
P
Grundy
S M
SM
Patel
S B
SB
D017398:Alternative Splicing / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D005786:Gene Expression Regulation; D007438:Introns / Q000235:genetics*; D008074:Lipoproteins / Q000032:analysis / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D019154:Protein Splicing / Q000235:genetics / Q000502:physiology; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology
D001055:Apolipoproteins B; D002352:Carrier Proteins; D008074:Lipoproteins; C087966:apolipoprotein B-27.6; C106092:microsomal triglyceride transfer protein