Apolipoprotein B (apoB) mRNA editing involves site-specific deamination of a cytidine at nucleotide 6666, converting a CAA glutamine codon to a UAA translation stop codon. Lipoprotein particles assembled by large (apoB100) or small (apoB48) translation products from unedited and edited mRNA (respectively), have different structural and functional properties which affect their metabolism. Of particular biomedical significance is the finding that B48-containing lipoprotein particles are rapidly cleared from the serum and are not metabolized to low density lipoprotein (LDL), an atherogenic disease risk factor. The broad goal of the proposed research is to evaluate how the mechanism of apoB mRNA editing might serve as a controlling point for tissue-specific and metabolically regulated lipoprotein production. The specific hypothesis tested is that the interactions of protein factors leading to the assembly of editosomes are dynamic, and can be modified by the cell to regulate the amount of apoB mRNA edited. Published and preliminary data suggest the involvement of five or more proteins in the editing process. Cytidine deaminase (27 kDa) and three RNA-binding proteins (66,44 & 40 kDa) are proposed to serve the respective roles of enzymatic C->U conversion and apoB mRNA editing site recognition. The participation and function of 240 and 49 kDa proteins are more speculative and lie largely in the ability of these proteins to enhance in vitro editing efficiency. Preliminary data demonstrate production of antibodies reactive with p240 and p44, cloning and sequencing of cDNAs for p44 and p27.
The Specific Aims propose antibody development and cDNA characterization for factors involved in apoB mRNA editing and transfection studies to evaluate the occurrence of each factor and its requirement in apoB mRNA editing. Antisense oligo nucleotide inhibition of translation and gene knockout in cultured cells will be used to confirm each factor's requirement. McArdle and HepG2 cells will be used as test systems for they represent respectively; (1) a system where editing activity is readily detectable but could be experimentally enhanced or diminished, and (2) a system where editing can not be detected but may be induced experimentally through single or multiple transfections. In vitro systems for apoB mRNA editing will complement transfection studies in assessing editing factor functions. The requirement of each factor in editing will be evaluated in terms of the consequence to in vitro editing activity of removing factors by immunodepletion and of adding recombinant factors back to the extracts. Proposed structural interactions, such as those between p27 and the RNA-binding protein, will be directly addressed in vitro using reversible chemical and photochemical cross-linking strategies in conjunction with western blot analysis of diagonal 2D gels and immunoadsorption assays. Through the proposed research, it should be possible to determine which proteins and interactions are necessary and sufficient for editing to occur, and which factors and interactions are involved in modulating the level of editing.
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