The C-to-U editing of apolipoprotein-B (apo-B) mRNA is catalyzed
by an enzyme complex that recognizes an 11-nt mooring sequence
downstream of the editing site. A minimal holoenzyme that edits
apo-B mRNA in vitro has been defined. This complex contains
apobec-1, the catalytic subunit, and apobec-1 complementation
factor (ACF), the RNA-binding subunit that binds to the mooring
sequence. Here, we show that ACF binds with high affinity to
single-stranded but not double-stranded apo-B mRNA. ACF contains
three nonidentical RNA recognition motifs (RRM) and a unique
C-terminal auxiliary domain. In many multi-RRM proteins, the
RRMs mediate RNA binding and an auxiliary domain functions in
protein–protein interactions. Here we show that ACF does
not fit this simple model. Based on deletion mutagenesis, the
RRMs in ACF are necessary but not sufficient for binding to
apo-B mRNA. Amino acids in the pre-RRM region are required for
complementing activity and RNA binding, but not for interaction
with apobec-1. The C-terminal 196 amino acids are not absolutely
essential for function. However, further deletion of an RG-rich
region from the auxiliary domain abolished complementing activity,
RNA binding, and apobec-1 interaction. The auxiliary domain
alone did not bind apobec-1. Although all three RRMs are required
for complementing activity and apobec-1 interaction, the individual
motifs contribute differently to RNA binding. Point mutations in
RRM1 or RRM2 decreased the Kd for apo-B
mRNA by two orders of magnitude whereas mutations in RRM3 reduced
binding affinity 13-fold. The pairwise expression of RRM1 with
RRM2 or RRM3 resulted in moderate affinity binding.