A sequence-specific RNA-binding protein complements apobec-1 To edit apolipoprotein B mRNA.
The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.
Mehta
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Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Driscoll
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D000071479:APOBEC-1 Deaminase; D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000235:genetics*; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D012333:RNA, Messenger; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase