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nucleolar localization signal or whether RNA-binding is required for the nucle-
olar localization. However, an ADAR2 mutant containing point mutations
within both dsRBDs displayed exclusion from the nucleolus indicating RNA-
binding was required for nucleolar localization (Dawson
, 2004). These
results suggest that ADAR2 activity is regulated through subcellular localization.
The crystal structure of the deaminase domain of ADAR2 revealed the
presence of a zinc ion in the catalytic center as had been proposed but in
addition, they found an inositol hexakisphosphate molecule buried in the core
of the enzyme (Macbeth
et al.
, 2005). The amino acid residues that coordinate
the inositol hexakisphosphate are conserved throughout the ADAR and
ADAT1 families, but absent from the ADAT2/3 family. The inositol hexaki-
sphosphate molecule stabilizes the ADAR2 protein structure and is essential for
catalytic activity.
Similar to ADAR1, there are different isoforms of ADAR2. Human
ADAR2 cloned from a brain cDNA library encoded two major isoforms which
differed by the inclusion or exclusion of an
et al.
Alu
cassette within the deaminase
domain (Gerber
, 1997). Characterization of the two ADAR2 isoforms
revealed that both had the same substrate specificity, but inclusion of the
et al.
Alu
-
containing exon led to a twofold reduction in ADAR2 activity on a
GluR-B R/G
site substrate
. Another major transcript was isolated that lacked the
last two amino acids and surprisingly this isoform was catalytically inactive
(Lai
in vitro
, 1997).
Rat ADAR2 is alternatively spliced at the amino terminus and the
inclusion of one exon of 47 bp alters the predicted reading frame of the protein
(Rueter
et al.
, 1999). Use of the canonical translation start site in the presence
of the 47 bp exon would generate a truncated protein lacking the dsRBDs and
the catalytic domain. The inclusion of this exon is dependent upon editing of an
adenosine residue at the
et al.
1 position of the 3 0 splice site, which mimics
the canonical AG acceptor site. Editing at this site occurs up to 30% in
ADAR2 pre-mRNA isolated from rat brain. Therefore, rat ADAR2 has evolved
a complex autoregulatory feedback, such that alternative splicing of its own
transcript is dependent on the presence of a functional ADAR2 protein
(Rueter
, 1999). ADAR2 autoediting also occurs in human cell lines, and
the level of autoediting and inclusion of the alternatively spliced exon have been
shown to correlate, such that higher expression of ADAR2 results in increased
inclusion of the 47 bp exon (Maas
et al.
, 2001). This editing is independent of
ADAR1 expression. The stem-loop structure surrounding the edited splice site
is created by interactions between intron 4 and the downstream exon. Despite it
containing an intervening loop of 1354 nucleotides, the paired regions are highly
conserved.
et al.
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