Biology Reference
In-Depth Information
V. RNA BINDING BY ADARS
ADAR belongs to a diverse group of proteins that have one or more copies of a
dsRBD of approximately 70 amino acids (reviewed in Fierro-Monti and
Mathews, 2000). Despite only a few residues being conserved, all dsRBDs
analyzed fold into the same secondary structure of
-helices and
-sheets,
organized as
-helices make contact with the
same face of the RNA. Other proteins with dsRBDs members include
1-
1-
2-
3-
2, where the
a
protein involved in mRNA transport; PKR, an interferon-inducible, RNA-
dependent protein kinase; and many proteins involved in the RNA interference
pathway which have domain arrangements resembling ribonuclease III, an RNA
nuclease.
The dsRBD-containing proteins do not exhibit sequence specificity, it is
the secondary structure of the A-form RNA that they recognize. A-form nucleic
acid differs from B-form in that the major groove is narrow and the minor groove
is wide and shallow. Highly sequence-specific interaction between proteins and
DNA occurs on the major grove; however, on A-form nucleic acid this is not
possible so the protein-RNA interactions occur with the 2-hydroxyl group of the
ribose sugar in the minor and are therefore not sequence specific.
However, the dsRBDs of ADAR2 do display a binding selectivity to the
transcripts they edit. Experiments on a short RNA encompassing the
Staufen
GluR-B Q/
R
site demonstrated that ADAR2 dsRBDs exhibit selective RNA binding to the
GluR-B Q/R
site and this was distinct from the binding site of a dsRBD from PKR
(Stephens
, 2004). The dsRBD occupies approximately 16 bp of dsRNA but
many can accommodate or prefer binding to substrates that contain bulges or
loops within the dsRNA. The individual dsRBDs of ADAR2 exhibit different
binding specificities when analyzed separately (Poulsen
et al.
,
2006). dsRBD1 preferentially binds to perfect duplex dsRNA located in the
stem-loop region, whereas dsRBD2 shows preference for dsRNA containing an
A-C mismatch which is usually located near to the editing site. Both dsRBDs are
required for efficient editing by ADAR2 (Stefl
et al.
, 2006; Stefl
et al.
, 2006).
In mammals, ADAR1 contains three dsRBDs, whereas ADAR2 and
ADAR3 both contain two dsRBDs. Recently, an ADAR was cloned from squid
that is an ortholog of ADAR2 (Palavicini
et al.
, 2009). It encodes three dsRBD;
however, the first dsRBD is in an alternatively spliced exon at the amino
terminus of the protein. When both isoforms were expressed and assayed
in vitro
et al.
the isoform with three dsRBDs had increased enzymatic activity.
To investigate what was responsible for the specificity of ADAR1 and
ADAR2, chimeric proteins were generated with the dsRBDs from one ADAR
and the deaminase domain from another (Wong
, 2001). This revealed that
the editing specificity in the chimeric proteins was provided by the deaminase
domain. However, when the dsRBDs of ADAR1 and PKR were exchanged, this
et al.
