Abstract
An RNA editing mechanism converts C to U at nucleotide 6666 in apolipoprotein 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. F., and Davidson, N. O. (1993) Science 260, 1816-1819). In this study, we expressed p27 in McArdle 7777 cells, which edit apoB mRNA with low efficiency. The levels of editing enzyme increased 10-fold, and editing of the endogenous apoB mRNA increased 2-fold in p27-transfected cells. These results demonstrate that p27 is involved in editing in mammalian cells. p27 was also expressed in COS cells, which do not synthesize apoB and lack editing activity. Extracts from p27-transfected cells acquired the ability to edit apoB mRNA only when extracts from other tissues were added. This reconstituted enzyme had the same sequence specificity as the native enzyme. The activity that complemented p27 function was detected in baboon liver and in tissues that do not synthesize apoB, including kidney and testis. The COS expression system was used to analyze the structure and function of p27, which contains a putative zinc finger (His61, Cys93, and Cys96) similar to other cytidine deaminases. Site-directed mutagenesis of His61 to Arg, Pro92 to Leu, or Cys96 to Ser abolished p27 activity. The zinc finger in p27 may be required for catalysis, protein-protein interactions, or binding to apoB mRNA.
Highlights
RESULTSThis construct was used in transient transfection p27 protein was expressed, we added extracts from the transstudies of McArdle 7777 cells, a rat hepatoma cell line that fected cells, referred to as p27/COS, to extracts from baboon synthesizes both apoBlOO and apoB48 mRNA
“he levels of editing enzyme increased 10-fold, anded- kDa, p27, which appears to be the catalytic subunit of the iting of the endogenous apoBmRNA increased 2-foldin editing enzyme (12). p27 contains a putative zinc finger of 3
Extractsfrom p27-trans- When expressed in Xenopus oocytes, p27 was capable of deamifected cells acquiredthe ability to edit Apolipoprotein B (apoB) mRNAonly nating cytidine but lacked the ability to edit apoB mRNA in when extracts from other tissues were added
Summary
This construct was used in transient transfection p27 protein was expressed, we added extracts from the transstudies of McArdle 7777 cells, a rat hepatoma cell line that fected cells, referred to as p27/COS, to extracts from baboon synthesizes both apoBlOO and apoB48 mRNA. These results demonstrate thattransfected COS and the cells were transfected, the effects of the overexpression of CHO cells express p27 but lack additional proteins that are p27 on editing in McArdle 7777 cells will be underestimated. McArdle 7777 cells contain low levels of editing activity (0.35 from baboon liver, a tissue that synthesizes apoB but lacks pg of RNA editedpg of protein), there waas 10-fold increase in editing activity (9). Mixing experiments showed that that p27 expressed in Xenopus oocytes lacked the ability to editthe lack of activity in these extracwtsas not duteo the presence apoB mRNA in vitro unless extracts from other tissues were of a n inhibitor (Ref. 9 and datanot shown)
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