A. Mehta et Dm. Driscoll, A SEQUENCE-SPECIFIC RNA-BINDING PROTEIN COMPLEMENTS APOBEC-1 TO EDIT APOLIPOPROTEIN-B MESSENGER-RNA, Molecular and cellular biology, 18(8), 1998, pp. 4426-4432
The editing of apolipoprotein B (apo-B) mRNA involves the site-specifi
c deamination of cytidine to uracil. The specificity of editing is con
ferred by an 11-nucleotide mooring sequence located downstream from th
e editing site. Apobec-1, the catalytic subunit of the editing enzyme,
requires additional proteins to edit apo-B mRNA in vitro, but the fun
ction of these additional factors, known as complementing activity, is
not known. Using RNA affinity chromatography, we show that the comple
menting 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 t
o apo-B mRNA but not to the triple-mutant RNA. This protein was not de
tected in the eluates from the mutant or the antisense RNA columns. In
troduction of the mooring sequence into luciferase RNA induced cross-l
inking of the 65-kDa protein. A 65-kDa protein that interacted with ap
obec-1 was also detected by far-Western analysis in the eluate from th
e wild-type RNA column but not from the mutant RNA column. For purific
ation, proteins were precleared on the mutant RNA column prior to chro
matography on the wild-type RNA column. Silver staining of the affinit
y-purified fraction detected a single prominent protein of 65 kDa. Our
results suggest that the complementing activity may function as the R
NA-binding subunit of the holoenzyme.