Cloning and mutagenesis of the rabbit ApoB mRNA editing protein. A zinc motif is essential for catalytic activity, and noncatalytic auxiliary factor(s) of the editing complex are widely distributed.
Apolipoprotein (apo) B mRNA editing is the specific deamination of cytidine (nucleotide 6666) to uridine in apoB mRNA. We isolated a full-length cDNA clone encoding the rabbit apoB mRNA editing protein (REPR), a subunit of the editing complex. Rabbit REPR is analogous to a rat enterocyte 27-kDa protein that has been shown to have cytidine deaminase activity. Like rat REPR, rabbit REPR edited synthetic apoB RNA when mixed with chicken enterocyte extract. Surprisingly, the REPR also acquired editing activity when mixed with extracts from various organs of the rabbit (liver, gallbladder, stomach, intestine, adrenals, thyroid, testes, spleen, kidney, and lung) or the chicken (kidney and liver). In contrast, the rabbit REPR mRNA was found only in the small and large intestine. Thus, the auxiliary protein(s) of the apoB mRNA editing complex, which are essential for editing activity, exist in organs devoid of significant apoB mRNA editing or apoB synthesis. REPR requires zinc for its catalytic activity. We mutated putative zinc-coordinating residues (His61, Cys93, Cys96) and 2 additional residues (Glu63, Pro92) of the rabbit REPR that are conserved in other cytidine or deoxycytidylate deaminases and in rat REPR. The wild-type and mutant REPR cDNAs each produced 28-kDa proteins when transcribed and translated in vitro. Compared with the wild-type editing activity, the mutations of His61-->Ala, Glu63-->Ala, Cys93-->Ala, and Cys96-->Ala abolished or greatly reduced editing activity, whereas the mutations of His61-->Cys (which also can coordinate zinc) and Pro92-->Ala had a lesser effect. These results indicate that His61, Cys93, and Cys96 are essential for editing activity, probably because they coordinate zinc, whereas Glu63 also is essential, because it may be involved in the deaminase reaction. In addition, the widespread distribution of the auxiliary factor(s) portends their involvement in other RNA editing reactions.
Yamanaka
S
S
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
Poksay
K S
KS
Balestra
M E
ME
Zeng
G Q
GQ
Innerarity
T L
TL
D000071479:APOBEC-1 Deaminase; D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000235:genetics*; D007413:Intestinal Mucosa / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017393:RNA Editing; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D015032:Zinc / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D001055:Apolipoproteins B; D011994:Recombinant Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase; D015032:Zinc