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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.
The Journal of Biological Chemistry
|September 15, 1989
Summary
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.