Apolipoprotein B (apo B) is an obligate structural component of both intestinal and hepatic triglyceride-rich lipoprotein and plays a central role in their catabolism. Two distinct isomorphs (apo B100 and apo B48) are synthesized, in a tissue-specific manner, from a common gene. Apo B48 is produced in mammalian small intestine and arises as a result of a site-specific, posttranscriptional, cytidine deamination of apo B m RNA, referred to as apo B mRNA editing. In this reaction, a CAA (glutamine) codon in apo B100 is edited to a UAA, stop codon and results in apo B48 synthesis. The truncated protein lacks the domains, present in apo B100, for binding to the LDL receptor and also for interaction with apo (a) which leads to the formation of Lp(a). As a result, apo B mRNA editing assumes central importance in determining the atherogenic potential of apo B. Apo B mRNA editing is mediated by protein factor(s), which together constitute the apo B RNA editing enzyme. One of the components, REPR, (rat apo B mRNA editing protein) has been cloned by the investigator, REPR is most likely the catalytic subunit of the apo B mRNA editing enzyme complex and has an absolute requirement for additional protein components [complementation factors] in order to mediate apo B RNA editing. However, there is little information concerning the nature and identity of these complementation factors. Additionally, although REPR has been shown to function as a component of the apo B mRNA editing enzyme through complementation and transfection studies, its role is not established to be indispensable. Accordingly, the objectives of this proposal are, first, to identify proteins which interact with REPR and complement apo B mRNA editing. In the second aim, we will mutate critical residues within a conserved Zn-binding domain in REPR, present in other cytidine deaminases. Using a functional OST-REPR fusion protein, we will determine the effects of these mutations on apo B RNA editing. Additionally, in studies which represent the major focus of this second aim, we will determine the biological importance of REPR to ap0 B gene expression. This will be achieved by means of targeted gene disruption both in ES cells and in selected cell lines, using genomic clones isolated and characterized by the investigator. This approach will allow a definitive assessment of the importance of REPR and its homologs in apo B mRNA editing and in the regulation of apo B gene expression. Taken together, this approach should allow elucidation of the molecular mechanisms underlying this novel pathway for gene regulation in the mammalian small intestine.
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