Partial characterization of the auxiliary factors involved in apolipoprotein B mRNA editing through APOBEC-1 affinity chromatography.
APOBEC-1-catalyzed apolipoprotein B (apoB) mRNA editing requires auxiliary factors, but the number and functions of these factors are unknown. We have partially purified the editing activity from extracts of a McArdle cell line overexpressing His6-hemagglutinin-tagged, rat APOBEC-1 using metal-chelating affinity chromatography. The 1,200-fold purification achieved by this approach was partially dependent on exogenously added RNA containing a mooring sequence for editosome assembly. Affinity-purified editing activity could be separated by 300 mM NaCl extraction into two fractions, a salt-resistant fraction (editing fraction 1; EF1) and a salt-soluble fraction (EF2). Neither EF1 nor EF2 alone could edit apoB RNA, but when added together they reconstituted full editing activity. Previously identified candidate auxiliary factors including the p66/p44 apoB RNA binding proteins and the presumptive editosome assembly factor p240 were all present in the affinity-purified editing complex. Moreover, virtually all of p66, p240, and APOBEC-1 were present in EF1, whereas p44 was quantitatively recovered in EF2. This is the first demonstration that p66 and p44 can bind to apoB RNA independently of one another. In addition, 100- and 55-kDa apoB RNA cross-linking proteins have been identified in the APOBEC-1 affinity-purified material. RNA competition studies demonstrated that p100, p66, and p55 bound selectively to apoB RNA, whereas p44 had general RNA cross-linking characteristics. The data underscore the multiplicity of auxiliary factors potentially involved in apoB RNA editing and suggest an editosome far more complicated than may have been previously appreciated.
Yang
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Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642, USA.
Yang
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Kovalski
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Smith
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HC
D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D003564:Cytidine Deaminase / Q000235:genetics*; D017393:RNA Editing; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D001055:Apolipoproteins B; D012333:RNA, Messenger; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase