Hepatic synthesis and secretion of apolipoprotein B (apo B) is metabolically regulated by dietary and hormonal factors. Recent evidence suggests that diet and hormones regulate a novel form of RNA processing known as mRNA editing. Edited apo B mRNA translates into a lower molecular weight protein (apo B -L) due to the conversion of a glutamine codon to an in-frame stop codon through a specific single base change of cytidine to uridine in codon 2153 (CAA->UAA). Macromolecular assemblies involved in mRNA editing (editosomes) have been recently identified in vitro and proposed to carry out the specific C->U conversion on riboprobes of apo B cDNA subclones. RNA editing and the 27S editosome have characteristic properties of recognition, assembly and catalysis seen with many other ribonucleoprotein-dependent processes. Immunological, molecular and biochemical techniques have been proposed for purifying editosomes, characterizing their macromolecular composition and determining the mechanism underlying RNA recognition and nucleotide conversion. Monoclonal antibodies specific for the editosome components will be produced and immunoassays developed for qualitative and quantitative analyses of structure and function. These studies will be complemented by molecular analysis of editing-specificity using mutant apo B mRNA constructs which focus on sequences flanking the editing site. Description of tissue-specific characteristics which might underlie the quantitative differences in editing activity seen in liver and intestine will also be evaluated. The significance of the proposed research lies in its potential to describe metabolic regulation of apo B production at the level of the mechanism of mRNA editing and the specific macromolecules whose synthesis and/or interactions participate in the control. The results of the proposed studies have implications regarding the hepatic production of LDL (unedited apo B) and intestinal production of chylomicrons (edited apo B) with respect to the variable atherogenic potential of these lipoprotein fractions in humans. Moreover, the information obtained in these studies will be important for evaluating the generic properties of mRNA editing and identifying other mRNAs which might serve as editing substrates.
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