Structure and Double-stranded RNA Cleavage Activity of the RNase
IIIb Domain of Human Dicer
Mary Myers '12 and Brian Thorne '12
Dicer protein is responsible for generating short, non-coding
microRNAs duplexes from
dsRNA, and is therefore critical for the
which leads to sequence-specific translational repression of genes.
duplexes formed via Dicer cleavage are generated with symmetric
2–3nt 3' overhangs and 5'-phosphate and 3'-hydroxyl groups,
which trigger the degradation of mRNAs with a matching
Human Dicer is a large, multi
Daltons in size, and is classified as a class 3 RNase
protein. Classification is based on
the observation of two RNase III nuclease
IIIa and RNase
of these domains can be found either as fragments, or
can coordinate into homodimers.
this tutorial focuses predominately on
the RNase IIIb
domain of the protein, the folllowing image represents the
included in a complete dicer complex:
has indicated that the nature of the single processing center in Human
(Hs)-Dicer, suggests that RNase IIIb domain fragments are capable of
dimers, which result in two catalytic sites within the processing
center; one on each end of the dimer interface.
phosphodiester bonds and generates ~20nt dsRNA
products with 2 nt 3' overhangs.
Other domains, such as the RNA Binding
domain (RBD), dsRNA Binding
Domain (dsRBD) and PAZ assist the cleavage reaction.
The RNase IIIb domains specifically requires Mg2+
catalyze the hydrolysis of the RNA phosphodiester backbone.
structure of RNase IIIb domain is
known to a resolution of 2.0
Å. Each of the monomers that constitute the RNase
IIIb homodimer possess a highly conserved structure with
contains 8 α-helices as
as a relatively rare 310
helix (helix 2),
ß-strands in its structure. The
dimerization of the
monomers and the protein's specific folding result in a negatively
charged valley which supports
the Mg2+ ions required for
cleavage. This negative protein valley
is represented in the
accompanying image by the blue region
within the interface of the
The charge difference within the valley provided by the
metal ions allows the dsRNA substate to bind more readily by possibly
neutralizing the repulsive charge, and in addition results
in the resolution of two
RNA cutting sites within
site. Two cutting
sites are necessary at each catalytic
site, so that both strands of the dsRNA substrate can be processed, and
the 3' overhang can be formed.
The RNase IIIb exists in solution as a stable dimer with
tight associations. The interface
between the two monomers forms the
base of the previously mentioned
via interactions between the anti-parallel
as well as interactions between the α7
Hydrophobic interactions are the main driving force
the interactions of aromatic residues from both monomers in the dimer
interface formed by the two
interacts with [Thr]1717,
interacts with [Ile]1711
The Leucine residues
increase dimer stability by associating the α9
the dimer interface.
interacts with [Arg]1703
near the ends of the α3
mentioned, two Mg2+ ions
are bound by each
IIIb domain either at, or close to, the active site.
two ions are differentiated as Mg-1
location within the active site. These four
ions are each
coordinated by eight interactions
Mg-1 atoms directly
interact with carboxylate
as three molecules of water,
interacts with one of the three coordinated water
stabilize the Mg-1
interacts directly with the carboxylate
oxygen of [Asp]1709
and four water
also interacts with one
of the water molecules here as well
residues (only one shown) positioned
centrally in the homodimer interact
with the coordinated water
is presumed that the Mg-1
functions to activate the
water molecule, and is positioned appropriately within the
it to serve this purpose.
near the end of the catalytic
site places the nucleophile in the
most stereochemically favorable area
within this specific domain. When complexed in a crystal with
enzyme, the dsRNA substrate ended up
colliding with the N-terminus
within the RNase IIIb domain.
this information, a
comformational change is likely to
occur either in the protein or with
the dsRNA substrate; maybe even both. When the substrate is
within the dimer interface, the Mg-2 ions are closely associated with
the minor groove of the RNA helix between the two cleavage sites.
A logical explanation for this observation would be to
the negatively charged valley within the dimer near the active sites.
 Cioca, DP et al. 2003.
RNA interference is the functional pathway with therapeutic
potential in huma myeloid leukemia cell lines. Gene
Cancer Therapy. Feb;
Takeshita, D. et al. 2007.
Double-stranded RNA Cleavage Activity of the C-terminal RNase III
Domain of Human Dicer. The
Journal of Molecular Biology
Blaszcyk, J. et al. 2004. Noncatalytic Assembly of Ribonuclease III
with Double-Stranded RNA. Structure
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