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Apolipoprotein B48 RNA editing in chimeric apolipoprotein EB mRNA
K Boström1, S J Lauer, K S Poksay
1Department of Physiology, Gladstone Foundation Laboratory for Cardiovascular Disease, University of California, San Francisco 94140-0608.
Abstract:
Apolipoprotein (apo) B occurs in two forms, apoB100 (512 kDa) and apoB48 (240 kDa); both are derived from the same gene. A novel mechanism involving editing of the apoB mRNA causes the formation of apoB48; the first base of codon 2153 is changed from cytosine to uracil, converting a glutamine codon to a premature stop codon. To identify the apoB mRNA sequence elements recognized by the apoB mRNA editing mechanism, two apoB cDNA fragments (354 and 63 base pairs) with codon 2153 near their centers were inserted into a high expression vector of another secreted apolipoprotein, apoE. The resulting vectors, pHEB-354 and -63, were transfected into Chinese hamster ovary cells, HepG2 cells, and apoB48-producing CaCo-2 cells. The secreted chimeric apolipoproteins (apoEB354 and apoEB63) were analyzed for premature truncation, and the mRNA was analyzed for the presence of an edited base. The pHEB-354 construct produced a truncated protein only in CaCo-2 cells, whereas pHEB-63 produced no truncated protein in any of the three cell types. The mRNA was converted to cDNA and amplified by the polymerase chain reaction technique. Differential hybridization of the polymerase chain reaction products with CAA (Gln) and TAA (Stop) specific probes detected an edited base only in cDNA from CaCo-2 cells transfected with pHEB-354, in agreement with the protein analysis. We conclude that the nucleotide sequence of the apoB cDNA insert in pHEB-354 contains sufficient information to be edited in CaCo-2 cells. In these cells, a cryptic polyadenylation site was activated in the edited pHEB-354 mRNA. As a result, CaCo-2 cells transfected with pHEB-354 produced a short, edited pHEB-354 mRNA and a long, unedited pHEB-354 mRNA. Chinese hamster ovary cells transfected with pHEB-354 or CaCo-2 cells transfected with pHEB-63 produced only a full length transcript. Amplification of the pHEB-354 cDNA using 3'-primers upstream and downstream of the poly(A) addition site and hybridization with the TAA probe confirmed these results. This unusual mRNA editing apparently occurs before polyadenylation, probably in the nucleus.
Insights
Researchers identified specific apoB mRNA sequence elements responsible for creating apoB48 through RNA editing. This editing mechanism, involving a base change, occurs before polyadenylation in CaCo-2 cells, leading to truncated apolipoprotein B production.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Apolipoprotein (apo) B exists as apoB100 and apoB48, derived from the same gene.
- ApoB48 formation results from a novel apoB mRNA editing mechanism, changing codon 2153 from glutamine to a premature stop codon.
Purpose of the Study:
- To identify the specific sequence elements within apoB mRNA that are recognized by the apoB mRNA editing machinery.
- To investigate the cellular context required for apoB mRNA editing and subsequent protein truncation.
Main Methods:
- Insertion of apoB cDNA fragments (354 and 63 bp) containing codon 2153 into an apoE expression vector.
- Transfection of these vectors into Chinese hamster ovary (CHO), HepG2, and CaCo-2 cells.
- Analysis of secreted chimeric apolipoproteins for premature truncation and mRNA for edited bases using polymerase chain reaction (PCR) and differential hybridization.
Main Results:
- The pHEB-354 construct yielded truncated protein only in CaCo-2 cells, while pHEB-63 showed no truncation in any cell type.
- PCR analysis confirmed edited bases only in CaCo-2 cells transfected with pHEB-354, correlating with protein truncation.
- In CaCo-2 cells, pHEB-354 mRNA editing activated a cryptic polyadenylation site, producing both edited (short) and unedited (long) transcripts.
Conclusions:
- The apoB cDNA sequence within pHEB-354 contains sufficient information for editing in CaCo-2 cells.
- This mRNA editing event appears to occur prior to polyadenylation, likely within the nucleus.
- The study elucidates key sequence elements and cellular conditions governing apoB mRNA editing for apoB48 production.