EXPRESSION AND CHARACTERIZATION OF P27, THE CATALYTIC SUBUNIT OF THE APOLIPOPROTEIN-B MESSENGER-RNA EDITING ENZYME

An RNA editing mechanism converts C to U at nucleotide 6666 in apolipo protein B (apoB) mRNA The catalytic subunit of the editing enzyme, p27 , was recently cloned. When expressed in Xenopus oocytes, p27 required other proteins to edit apoB mRNA in vitro (Teng, B., Burant, C. E, an d Davidson, N. O. (1993) Science 260, 1816-1819). In this study, we ex pressed p27 in McArdle 7777 cells, which edit apoB mRNA with low effic iency. The levels of editing enzyme increased 10-fold, and editing of the endogenous apoB mRNA increased 2-fold in p27-transfected cells. Th ese results demonstrate that p27 is involved in editing in mammalian c ells. p27 was also expressed in COS cells, which do not synthesize apo B and lack editing activity. Extracts from p27-transfected cells acqui red the ability to edit apoB mRNA only when extracts from other tissue s were added. This reconstituted enzyme had the same sequence specific ity as the native enzyme. The activity that complemented p27 function was detected in baboon liver and in tissues. that do not synthesize ap oB, including kidney and testis. The COS expression system was used to analyze the structure and function of p27, which contains a putative zinc finger (His(61), Cys(93), and Cys(96)) similar to other cytidine deaminases. Site-directed mutagenesis of His(61) to Arg, Pro(92) to Le u, or Cys(96) to Ser abolished p27 activity. The zinc finger in p27 ma y be required for catalysis, protein-protein interactions, or binding to apoB mRNA.