Expression and characterization of p27, the catalytic subunit of the apolipoprotein B mRNA editing enzyme.
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.
Driscoll
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Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195.
Zhang
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D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D002478:Cells, Cultured; D003564:Cytidine Deaminase / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D012323:RNA Processing, Post-Transcriptional; D051381:Rats; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014981:Xenopus; D016335:Zinc Fingers / Q000235:genetics; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D001055:Apolipoproteins B; D009838:Oligodeoxyribonucleotides; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase