ApoB mRNA editing is an exquisitely site specific RNA modification that results in C to U deamination of a single cytidine base and creates a translational stop codon. This process occurs in the mammalian small intestine and is mediated by a multicomponent enzyme complex containing apobec-1, as well as other protein components of unknown identity. Apobec-1 is an RNA binding protein with specificity for AU-rich RNAs. Furthermore, RNA binding activity of apobec-1 is required for C to U editing of apoB mRNA. Progress over the last period has led to the identification of a high affinity binding site present in a single copy in apoB RNA close to the edited base. This motif is present in the 3' untranslated region (UTR) of a number of oncogenes, including c-myc, and cytokine mRNAs, including TNFalpha, IL-2 and GM-CSF. Each of these mRNAs contains AU-rich elements, which are known to be key mediators of mRNA stability. Apobec-1 binds to these RNAs in vitro and in vivo, an effect associated with increased stability of c-myc mRNA. These data suggest that apobec-1 may regulate the expression of genes other than apoB. The investigators have further established that apobec-1 mediates C to U editing of NF1 mRNA, encoding the tumor suppressor, neurofibromin. Like apoB, NF1 RNA contains the consensus binding site in proximity to the edited C. C to U editing of NF1 mRNA, as in apoB mRNA, creates a translational stop codon and causes truncation of the protein. These findings serve as background for an examination of the mechanisms and requirements for apobec-1 mediated RNA binding and also for C to U editing of mRNAs, beyond apoB. Apobec-1 is required for C to U editing of apoB, but alone is insufficient, there being other proteins required. The number and identity of these factors is unknown. The investigators have used a two-hybrid screen to clone a candidate factor, p54, whose interactions, distribution and function make it a candidate apoB RNA editing associated protein. They will examine the question of what additional proteins are required for the assembly and function of apoB RNA editing enzyme, starting with a detailed characterization of p54. Finally, we have identified multiple apobec-1 related proteins (ARPs) in the human genome, using the signature sequence of apobec-1 flanking the catalytic site. One of these proteins, ARP-1 contains about40 percent homology to apobec-1 and demonstrates cytidine deaminase and apoB RNA binding activity. The investigators will examine the hypothesis in the current application that there are other cytidine deaminases, related to apobec-1, with functions involving site-specific RNA interactions.
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