Our long term goal is to understand the process of mRNA editing as a novel mechanism for regulating gene expression.
The aim of this proposal is to investigate the editing of apolipoprotein-B (apo-B) mRNA. Apo-B mRNA is modified in the mammalian intestine by a mechanism that converts a cytosine at nucleotide 6666 to a uracil. We developed an in vitro assay for the editing of apo-B mRNA which has been used to identify the mRNA sequences and cellular factors that are required for this reaction. The editing of apo-B mRNA is mediated by a site-specific cytidine deaminase that recognizes an II nucleotide sequence downstream from the editing site. The cDNA that encodes the catalytic subunit of the enzyme, p27, was recently cloned. p27 shares limited homology with the active sites of other cytidine deaminases, but requires other protein(s) to specifically edit apo-B mRNA. The activity that complements p27 has not been identified, but it may represent the RNA-binding subunit of the enzyme. We have detected the complementing protein(s) in tissues that do not synthesize p27 or apo-B, which suggests that this activity may be involved in other RNA editing or RNA processing events.
The specific aims are to: 1) Analyze the function and expression of p27. The activities of wild type and mutant p27 proteins will be analyzed using in vitro assays for editing, catalysis, RNA binding, zinc coordination, dimerization, and interaction with the other subunit(s) of the editing enzyme. 2) Identify and characterize the complementing protein(s). We have partially purified the complementing protein(s) from baboon kidney extracts. Preliminary experiments suggest that this activity is a 65 kDa protein or protein complex which can interact with p27 in vitro. The complementing activity will be purified to homogeneity by protein-affinity or RNA-affinity chromatography. Four strategies are proposed to isolate cDNA clones that encode the complementing protein(s). Using purified components of the editing reaction, the functions of the complementing activity and p27 will be studied in vitro. 3) Identify other mRNAs which undergo editing. We hypothesize that the editing enzyme recognizes both sequence and structure in apo-B mRNA. We propose to determine the secondary structure of apo-B mRNA in the region of the editing site. Other mRNA targets of the enzyme or its subunits will be identified using tissue-specific combinatorial RNA shape libraries. The results of our studies will provide insight into the mechanism of apo-B mRNA editing.
