Abstract
The messenger RNA for apolipoprotein B undergoes a discrete and specific C to U editing of nucleotide 6666. This generates a stop translation codon and defines the carboxyl terminus of apolipoprotein B48. A 27-kDa rat intestinal protein that does not itself edit apolipoprotein B mRNA, but confers editing activity on chick intestinal extracts that do not have intrinsic editing activity, has recently been identified and its cDNA cloned (Teng, B., Burant, C. F., and Davidson, N. O. (1993) Science 260, 1816-1819). Here we show that p27 is homologous in the zinc coordinating region of the active site to cytidine deaminases from Escherichia coli, Bacillus subtilis, yeast, and man and to deoxycytidylate deaminases from T2 and T4 bacteriophages and man. p27 expressed in Xenopus laevis oocyte extracts has cytidine deaminase activity and specifically confers editing activity on chick intestinal extracts. The homologous E. coli cytidine deaminase does not confer editing activity. The zinc-specific chelating agent o-phenanthroline abolishes p27 activity and site-specific apolipoprotein B mRNA editing in rat enterocyte editing extracts. We conclude that p27 is the catalytic subunit of the apolipoprotein B mRNA editing enzyme and is a zinc-containing cytidine deaminase.
Highlights
With singleor multiple added or deleted U residues by reverse transesterification
More subtle forms of mRNA editing involve the alteration of specific nucleotides in the mRNA sequence
A 27-kDa rat geted single A residues to form I in double-stranded RNA (12, intestinal protein that does not itself edit apolipopro- 13).Most plausibly, the editingof apoB mRNA is by the process tein B mRNA, but confers editing activity on chick in- of site-specific cytidine deamination
Summary
Data Base Searchesand Alignments-Homology searches and alignments were performed using the Multiple Alignment Constructionand Analysis Workbench (MACAW Version 1.05 for Windows) [19].All relevant sequences availablein the GenBanWEMBL data bases were retrieved and translated to the corresponding peptide sequence. Oocyte Expression“p27 mRNA expression and extract preparation were exactly as described by Teng et al [18]. Cytidine Deaminase Assay-A spectrophotometric assay was based on the method of Wang et al [20].The assay was performed in a total volume of 70 pl, using a l-cm microcuvette, and monitoredat 286 nm. The assay was carried out in the presence of 0.2-1 mg/ml celel xtract, mM Tris-HC1, pH 7.4,and 0.3m~ cytidine. A cloned cytidine deaminase from Escherichia coli (EC3.5.4.5)was used as a positive control [21]. RNA Synthesis-The DNA templates and methods used to prepare unlabeled RNA substrate and mutant substrate RNA havebeen described [9, 15, 22].
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