TY - JOUR
T1 - Activation of a cryptic splice-site in intron 24 leads to the formation of apolipoprotein B-27.6
AU - Nemeth-Slany, Attilla
AU - Talmud, Phillipa
AU - Grundy, Scott M
AU - Patel, Shailendra B.
N1 - Funding Information:
We gratefully acknowledge the gifts of monoclonal antibody C1.4 from Dr Gustav Schonfeld, to Dr Reca Infante for supplying the DNA for HMZ originally, to Loyce Rutledge for technical assistance and to Dr Helen Hobbs for critical reading of this manuscript. ANS was funded by a scholarship from the Szechenyi Istvan Foundation of Hungary. PJT is supported by the British Heart Foundation (RG 95-007). This work was supported by unrestricted grants from Merck, West Point, PA; Bristol Myers Squibb, New Brunswick, NJ; the Southwestern Medical Foundation, Dallas, TX and the Moss Heart Foundation, Dallas TX and by NIH Grant HL-29252.
PY - 1997/9
Y1 - 1997/9
N2 - 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;36:20902103). 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.
AB - 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;36:20902103). 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.
KW - Hypobetalipoproteinemia
KW - Microsomal triglyceride transfer protein
KW - Splicing
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U2 - 10.1016/S0021-9150(97)00105-6
DO - 10.1016/S0021-9150(97)00105-6
M3 - Article
C2 - 9298676
AN - SCOPUS:0030779051
SN - 0021-9150
VL - 133
SP - 163
EP - 170
JO - Atherosclerosis
JF - Atherosclerosis
IS - 2
ER -