Base modification editing of mRNAs involves the deamination of single nucleotides (A->l or C->U) in the coding region of the transcript, which generates alternative mRNAs that encode proteins with different biological functions. This proposal focuses on the mechanism of C->U editing in a mammalian system. The editing of apolipoprotein-B (apo-B) mRNA involves the deamination of C6666 to U which converts a glutamine codon (CAA) to an in-frame stop codon (UAA). The full-length and truncated apo-B proteins have distinct functions in lipoprotein metabolism and atherosclerosis susceptibility. Apo-B mRNA editing is catalyzed by a multi-protein complex which recognizes a nucleotide mooring sequence downstream of the editing site. The catalytic subunit of this complex, apobec-1, has cytidine deaminase activity but requires additional proteins to edit apo-B mRNA. We purified a protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The Apobec-1 Complementation Factor (ACF) cDNA encodes a novel 64.3 kDa protein that interacts with apobec-1 and binds to apo-B mRNA in a mooring sequence-specific manner. ACF and apobec-1 comprise the minimal protein requirements for editing of apo-B mRNA in vitro. We also have evidence which suggests that ACF is involved in editing in vivo . Our results support a model of the editing enzyme in which ACF functions as the RNA-binding subunit that docks apobec-1 to deaminate the upstream C.
The aims of this propsal are to: 1) Analyze the function of ACF in editing apo-B mRNA. We propose to identify the domains in ACF that are required for the editing of apo-B mRNA in vitro, test the hypothesis that ACF targets the editing enzyme to the nucleus, and test the hypothesis that ACF is required for apo-B mRNA editing in cells. 2) Characterize the holoenzyme complex. Our ability to reconstitute editing in vitro using recombinant proteins will allow us to investigate isolated steps in the editing reaction, including interaction of the enzyme subunits, binding of the enzyme to apo-B mRNA, and catalysis. We also plan to analyze complex formation in vivo and generate mutant enzymes defective in specific activities to analyze the functions of ACF and apobec-1 in the context of the holoenzyme. 3) Identify novel mRNA targets of ACF. ACF is widely expressed in tissues that lack apobec-1 and apo-B mRNA, which suggests that ACF may have additional functions in vivo. We plan to identify novel mRNA targets of ACF based on their homology with the ACF consensus binding site, their association with ACF in vivo, or their selection by ACF in vitro. The successful pursuit of these aims should provide insight into the mechanism of apo-B mRNA editing and the function of ACF.
