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Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts
1Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, Texas 78228-0147.
Abstract:
Apolipoprotein B (apoB) circulates in human plasma as two isoforms, apoB-100 (512 kDa) and apoB-48 (242 kDa). ApoB-48 is generated by a novel RNA editing mechanism which post-transcriptionally modifies apoB mRNA in the intestine by converting cytidine at nucleotide 6666 to uridine. This converts codon 2153 from glutamine (CAA) to a premature stop codon (UAA). To characterize the activity which edits apoB mRNA, extracts were prepared from enterocytes isolated from baboon small intestine. These extracts efficiently edit synthetic apoB RNA in vitro. Editing was detected by primer extension, and the specificity of the reaction was confirmed by DNA sequencing. Extracts prepared from other baboon tissues did not edit apoB RNA in vitro. The editing activity was partially purified by chromatography of the enterocyte extracts on DEAE-cellulose. The activity is sensitive to proteinase K but resistant to micrococcal nuclease and has an average molecular mass of 125 kDa when analyzed by gel filtration chromatography.
Insights
Researchers identified the RNA editing activity responsible for creating apolipoprotein B-48 (apoB-48) in baboon intestinal cells. This novel mechanism modifies apolipoprotein B mRNA (apoB mRNA) through a specific enzymatic process.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Apolipoprotein B (apoB) exists as two isoforms: apoB-100 and apoB-48.
- ApoB-48 is produced through a unique RNA editing process in the intestine.
- This process involves a specific cytidine to uridine conversion in the apoB mRNA.
Purpose of the Study:
- To characterize the enzymatic activity responsible for apoB mRNA editing.
- To investigate the tissue-specific nature of this RNA editing mechanism.
Main Methods:
- Preparation of extracts from baboon enterocytes and other tissues.
- In vitro editing assays using synthetic apoB RNA.
- Detection of editing by primer extension and DNA sequencing.
- Partial purification of the editing activity using DEAE-cellulose chromatography and gel filtration.
Main Results:
- Baboon enterocyte extracts efficiently edited synthetic apoB RNA in vitro.
- Editing activity was specific to intestinal extracts, not found in other tissues.
- The editing activity was sensitive to proteinase K and resistant to micrococcal nuclease.
- The enzyme responsible for editing has an estimated molecular mass of 125 kDa.
Conclusions:
- A novel RNA editing activity responsible for apoB-48 production has been identified and partially characterized.
- This activity is specific to intestinal cells and involves a protein-based enzyme.
- Further characterization of this enzyme could provide insights into gene expression regulation.