Related Experiment Videos
Sequence elements required for apolipoprotein B mRNA editing enhancement activity from chicken enterocytes
1Institute of Molecular Medicine, University of Texas-Houston, Houston, Texas, 77030, USA.
Abstract:
Mammalian intestinal apolipoprotein B (apoB) mRNA edits codon 2153 from CAA in apoB100 mRNA to a stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB mRNA contains a CAA codon at the corresponding site, but is not edited. Chicken enterocyte S100 extracts fail to edit mammalian apoB RNA, but contain factor(s) which enhance the mammalian enterocytes editing activity. By converting the chicken apoB mooring sequences to the conserved mammalian sequences, the study confirmed that this 11-nucleotide stretch was necessary and sufficient for minimal RNA editing. Using rat and chicken apoB chimeric constructs, the study revealed that mammalian apoB sequences were required for editing enhancement. In concert with the 29-nucleotide conserved cassette, the 5' rat apoB element (nucleotides 6615-6629) increased editing at C-6666, and was necessary for editing enhancement of chicken enterocyte S100 extracts. Similarly, the 3' rat apoB element (nucleotides 6726-6752) was required for editing enhancement of chicken enterocyte S100 extracts, but to a lesser extent in efficiency, compared to the 5' region. In conclusion, this study identified the sequences required for editing enhancement activity from chicken enterocyte S100 extracts.
Insights
Chicken intestinal cells lack apolipoprotein B (apoB) mRNA editing, unlike mammals. This study identified specific mammalian apoB sequences essential for enhancing this crucial RNA editing process in chicken cells.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Apolipoprotein B (apoB) mRNA undergoes a unique editing event in mammalian intestines, converting a glutamine codon (CAA) to a stop codon (UAA), resulting in apoB48 synthesis.
- This specific RNA editing is absent in chicken intestinal cells, despite the presence of a similar codon at the homologous site.
Purpose of the Study:
- To investigate the molecular basis for the species-specific difference in apoB mRNA editing between mammals and chickens.
- To identify the cis-acting sequences within apoB mRNA responsible for conferring editing activity and enhancing editing efficiency.
Main Methods:
- Construction and analysis of chimeric apoB mRNA constructs using rat and chicken sequences.
- Assays using chicken enterocyte S100 extracts to evaluate editing enhancement activity.
- Site-directed mutagenesis and sequence conversion to pinpoint essential regulatory elements.
Main Results:
- Conversion of chicken apoB mooring sequences to mammalian sequences restored minimal RNA editing, indicating the importance of this region.
- Mammalian apoB sequences were found to be necessary for enhancing editing activity in chicken enterocyte extracts.
- Specific 5' and 3' rat apoB elements were identified as crucial for enhancing editing efficiency, with the 5' element showing greater impact.
Conclusions:
- The study successfully identified specific mammalian apoB mRNA sequences that are necessary and sufficient for enhancing RNA editing.
- These findings elucidate the sequence requirements for apoB mRNA editing enhancement, contributing to the understanding of post-transcriptional gene regulation.