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resulted in a reduction or a complete loss of enzymatic activity on edited
transcript; however, the chimeric protein was still active on dsRNA. Therefore,
specificity is also provided by the dsRBDs (Liu
, 2000). ADAR3 is the only
member of the ADAR family known to bind to ssRNA, and experiments have
shown this interaction is mediated through the arginine- and lysine-rich R
domain in the amino terminus of the protein which is not found in other
ADAR proteins (Chen
et al.
, 2000; Fig. 3.1). ADAR3 is also capable of binding
dsRNA through its canonical dsRBDs but it is unable to edit either known
substrates or dsRNA.
The dsRBDs have additional roles other than binding dsRNA such as
nucleocytoplasmic shuttling of ADAR1 as previously described. The deletion of
dsRBD1 of ADAR2 reduced editing efficiency, whereas loss of dsRBD2 abolished
editing altogether (Poulsen
et al.
, 2006). The N-terminal region of ADAR2 had
an autoinhibitory effect on catalytic activity as a truncated ADAR2 protein
containing a deaminase domain and dsRB2 was capable of editing a 15-bp
substrate, whereas the full-length protein did not. However, the addition of the
N-terminal region of ADAR2 to the truncated protein in
et al.
reduced editing
efficiency on shorter substrates, suggesting that the RNA substrate has to be long
enough for binding of both dsRBDs to relieve the autoinhibition for efficient
editing (Macbeth
trans
, 2004).
The dsRBDs have also been implicated in dimer formation as binding to
RNA has been shown to be a prerequisite for dimer formation. However, there
have been conflicting reports and the issue of whether dimer formation requires
RNA binding for its formation has not been resolved. Studies have shown that
the formation of homodimers is required for ADAR activity (Cho
et al.
et al.
, 2003;
Gallo
, 2006). A ternary complex
can be observed when increasing amounts of ADAR are added to substrate RNA,
such that one monomer binds and then another, indicating dimerization is
RNA-dependent (Jaikaran
et al.
, 2003; Jaikaran
et al.
, 2002; Poulsen
et al.
using
one wild-type monomer and one catalytically inactive monomer showed that
both monomers contribute to hyperediting of dsRNA and site-specific editing of
substrates (Cho
et al.
, 2002). Analysis of RNA editing
in vitro
, 2003). However, fluorescence energy resonance transfer
(FRET) experiments indicate that ADAR1 and ADAR2 form homodimers in an
RNA-independent manner, and are capable of forming heterodimers
et al.
in vivo
(Chilibeck
, 2006).
To assess the role of RNA binding in dimerization (Valente and
Nishikura, 2007), mutations were made in three conserved lysine residues
(KKxxK
et al.
EAxxA) within each of the dsRBDs of ADAR1 and ADAR2. The
mutations introduced were based on data from alanine-scanning mutagenesis of
the
!
, which demonstrated that mutation
of exposed lysine residues within the conserved dsRBD eliminated RNA binding
without disrupting structure (Ramos
Drosophila
RNA-binding protein
Staufen
et al.
, 2000). Sequential purification of
