Structure, Vol. 13, 1403–1408, October, 2005, Published by Elsevier Ltd DOI 10.1016/j.str.2005.08.
005
Structure and Function
of Argonaute Proteins
Traci M. Tanaka Hall1,*
Laboratory of Structural Biology
National Institute of Environmental Health Sciences
National Institutes of Health
Research Triangle Park, North Carolina 27709
Summary
Argonaute (Ago) family proteins are multidomain pro-
teins expressed in prokaryotic and eukaryotic organ-
isms. In eukaryotes, Ago proteins are most well known
for their roles in RNA silencing. In prokaryotes, the
functions of Ago proteins are unknown, but based on
their similarity to eukaryotic Ago proteins, they could
be involved in nucleic acid-directed regulatory path-
ways related to RNA silencing. Recent structural and
biochemical studies have shed new light on the func-
tion of this family of proteins. These studies reveal how
these proteins recognize and cleave RNA and suggest
a function for prokaryotic family members.
Introduction
RNA interference, or RNAi, seems on its way to becom-
ing a household term. RNAi refers to a gene regulation
pathway in animals that “silences” the expression of a
target gene with sequences corresponding to the
double-stranded RNA that triggers the pathway. RNAi
was first reported in 1995 (Guo and Kemphues, 1995)
and similar pathways collectively referred to as RNA
silencing were discovered in plants (posttranscriptional
gene silencing) and fungi (quelling) earlier, reviewed in
Matzke and Matzke (2004). These early observations
noted unexpected silencing of gene expression when
exogenous RNA was introduced into organisms. The
beginning of our understanding of the mechanism of
RNA silencing began only 7 years ago in 1998 with the
experiments of Fire, Mello, and colleagues demonstrat-
ing that double-stranded RNA (dsRNA) triggered RNAi
(Fire et al., 1998). The application of RNA silencing has
been rapid, with research labs using the technology to
examine the effects of turning off genes of interest. The
promise of the use of RNAi as a therapeutic and diag-
nostic tool is tantalizing, taking RNAi to the headlines.
RNA silencing pathways process long dsRNAs into
small RNA molecules that direct the repression of tran-
scription or translation of nucleic acid targets with se-
quences corresponding to the small RNAs (Tomari and
Zamore, 2005, and references therein). Dicer family
RNase III enzymes cleave the dsRNA triggers of RNA
silencing pathways to produce small RNAs of w21 nt.
Long dsRNAs are processed to small interfering RNAs
(siRNAs) and endogenous stem-loop RNAs are pro-
cessed to microRNAs (miRNAs). Single strands of
siRNA and miRNA duplexes, referred to as guide strands,
*Correspondence: hall4@niehs.nih.gov
1
Lab address: http://dir.niehs.nih.gov/dirlsb/msghome.htm
Minireview
are incorporated into RNA silencing effector complexes
such as the RNA-induced silencing complex (RISC),
which cleaves mRNAs or represses their translation, or
the RNA-induced initiation of transcriptional gene si-
lencing (RITS) complex that regulates heterochromatin
assembly. These RNA silencing effector complexes
contain Ago family proteins.
Eukaryotic Ago proteins are characterized by two do-
mains, the PAZ and Piwi domains. PAZ domains are
small w140 residue domains found in Ago proteins and
Dicer enzymes. Structures of isolated PAZ domains
from Ago proteins alone or in complex with RNA have
been determined by X-ray crystallography and NMR.
The first structures of PAZ domains alone revealed that
the PAZ domain is a modified oligonucleotide/oligosac-
charide binding (OB) fold and suggested that the PAZ
domain may be responsible for binding to the 3# ends
of the siRNA (Lingel et al., 2003; Song et al., 2003; Yan
et al., 2003). Later structures of PAZ domains with RNA
representing the 3# overhanging ends of siRNAs il-
lustrated the mode of recognition of this structural fea-
ture of siRNAs (Lingel et al., 2004; Ma et al., 2004).
Piwi domains are found only in Ago proteins, and
their function was not known, as sequence alignment
did not reveal similarity to other proteins. The identity of
the component of RISC that cleaves or “slices” mRNA
targets remained elusive. Purification of minimized
cleavage-competent RISC suggested that the Ago pro-
teins were the major component and could be the cata-
lytic subunit of this effector complex that can cleave
target mRNAs as directed by the incorporated siRNA
or miRNA (Martinez et al., 2002). However, some of the
most illuminating evidence that Ago is the elusive Slicer
came from the crystal structure of an Ago protein.
With difficulties expressing sufficient amounts of full-
length eukaryotic Ago proteins, structural biologists
turned fruitfully to prokaryotic homologs with unknown
functions. The crystal structure of a full-length Ago pro-
tein from Pyrococcus furiosus (PfAgo) revealed that the
Piwi domain of Ago proteins is structurally related to the
RNase H family of ribonucleases (Song et al., 2004). This
protein comprises four major domains: N-terminal, PAZ,
middle, and Piwi domains. Structure-directed mutagene-
sis experiments implicate the Ago proteins as the cata-
lytic unit of the RISC (Liu et al., 2004). Similarly, the crystal
structure of a simpler archaeal Piwi protein from Archae-
oglobus fulgidus (AfPiwi) exposed the similarity to RNase
H (Parker et al., 2004). This protein retains the middle (A
domain) and RNase H-like Piwi (B domain) domains from
full-length Ago proteins but lacks the N-terminal and PAZ
domains. Very recently, a crystal structure of a full-length
eubacterial Ago protein from Aquifex aeolicus (AaAgo)
was determined and further confirmed the relationship to
RNase H (Yuan et al., 2005).
Several recent papers studying the structure and
function of Ago proteins have added to our understand-
ing of RNA recognition and cleavage by this family of
proteins (Ma et al., 2005; Parker et al., 2005; Rivas et
al., 2005; Yuan et al., 2005). Crystal structures of AfPiwi
in complex with siRNA mimics representing the 5# end
Structure
1404
of a guide siRNA bound to a target RNA have been
determined by two groups (Ma et al., 2005; Parker et
al., 2005). These structures provide insight into the re-
cognition of this end of the siRNA and its base pairing
with a target mRNA, and accompanying biochemical
work translates the findings to mammalian Ago pro-
teins. In addition, the crystal structure of PfAgo soaked
with manganese identified the active site residues in
Ago proteins (Rivas et al., 2005), and crystal structures
of an RNase H in complex with its substrate DNA:RNA
hybrid (Nowotny et al., 2005) suggest the conservation
of catalytic function in the RNase H enzyme family. Bio-
chemical work with a bacterially expressed human Ago
2 (hAgo2) protein fused with glutathione S-transferase
(GST) demonstrates that this protein with a single-
stranded guide siRNA forms a minimal recombinant
RISC with properties similar to RISC purified from
eukaryotic cells (Rivas et al., 2005). And the structure
of AaAgo also revealed that it may function as a DNA-
directed ribonuclease (Yuan et al., 2005), the first sug-
gestion of a function for these prokaryotic Ago proteins.
5ⴕ End Recognition by Argonaute Proteins
For several reasons, the 5# ends of siRNAs and miRNAs
are important for mRNA target recognition and definition
of the site of RNA cleavage. (1) Positions 2–7 of miRNAs
form the critical “seed” for target recognition. Base pair-
ing in this region is most important for target prediction/
effective translational repression by miRNAs (Enright et
al., 2003; Lewis et al., 2003, 2005; Stark et al., 2003;
Doench and Sharp, 2004; Haley and Zamore, 2004; Bren-
necke et al., 2005; Krek et al., 2005; Lim et al., 2005). (2)
A phosphate group at the 5# end of the siRNA strand that
forms the guide for mRNA target recognition has been
shown to be required for efficient RNAi (Nykanen et al.,
2001; Schwarz et al., 2002). (3) The substrate mRNA
cleavage site is directed by the distance from the 5# end
of the siRNA and occurs at the phosphodiester bond be-
tween the bases complementary to the 10th and 11th
bases of the guide siRNA (Elbashir et al., 2001a, 2001b;
Haley and Zamore, 2004; Martinez and Tuschl, 2004).
The importance of the 5# phosphate group also holds
true for recombinant RISC comprising a bacterially ex-
pressed GST-hAgo2 protein and a single-stranded
guide siRNA (Rivas et al., 2005). The 5# phosphate group
is important for the stability of the complex. Although
an active RISC can be formed with siRNA lacking a 5#
phosphate, less recombinant RISC is formed and non-
phosphorylated siRNA can be exchanged with compet-
itor phosphorylated siRNA. In contrast, phosphorylated
siRNA loaded into recombinant RISC is not exchanged
with competitor phosphorylated siRNA.
In addition, the 5# phosphate may be important for
the fidelity of the position of mRNA cleavage (Rivas et
al., 2005). GST-hAgo2 protein, like endogenous RISC,
cleaves target RNA between the bases complementary
to the 10th and 11th positions of the guide siRNA. If a
phosphorothioate is substituted at the cleavage site,
the target is cleaved poorly by recombinant RISC pro-
grammed with a phosphorylated guide siRNA in the
presence of Mg2+. If the reaction is performed in the
presence of Mn2+, the cleavage is partially rescued, as
had been observed for endogenous RISC (Schwarz et al.,
Figure 1. 5# Phosphate Binding Pocket of AfPiwi
A divalent metal ion (M) is coordinated by the 5# phosphate group
(5# P), Q159, the terminal carboxylate of L427, the third phosphate
group of the guide RNA (yellow), and a water molecule (wat). Black
dashed lines, metal coordination; red dashed lines, hydrogen
bonds. (AfPiwi, PDB code 2BGG.)
All figures were prepared with PyMol (http://www.pymol.org).
2004). However, if the same experiments are performed
with recombinant RISC programmed with nonphos-
phorylated guide siRNA, the phosphorothioate-substi-
tuted substrate is cleaved but at the phosphodiester
bond just 5# of the phosphorothioate. Thus, in the ab-
sence of the 5# phosphate, the guide siRNA can shift
within the enzyme to position a different phosphodies-
ter bond at the active site.
The crystal structures of AfPiwi with siRNA-like du-
plexes have now provided structural perspective on the
importance of the 5# end (Ma et al., 2005; Parker et al.,
2005). In both structures, the 5# nucleotide of the guide
siRNA is unpaired and bound in a pocket made up pri-
marily of residues in the A or middle domain. The first
base, U1 or A1, forms a stacking interaction with Y123,
a conserved aromatic position in Ago proteins (Figure
1). The unpairing of the first base explains its relative
unimportance for specifying miRNA targets (Enright et
al., 2003; Lewis et al., 2003, 2005; Stark et al., 2003;
Doench and Sharp, 2004; Haley and Zamore, 2004;
Brennecke et al., 2005; Krek et al., 2005; Lim et al.,
2005). The base-stacking interaction with the uracil or
adenine at the first position in the two structures sug-
gests that binding is sequence independent, but the
main chain nitrogen of N119 and the side chain oxygen
of T120 are observed to form direct (adenosine) or
water-mediated (uracil) hydrogen bonds with the first
base. This position is frequently a uracil in human miRNAs
and miRNAs from other species (Griffiths-Jones, 2004).
It seems unlikely from the crystal structures that bind-
ing by an Ago protein explains this conservation. T120
is somewhat conserved in human Ago proteins, but this
region is also variable, especially in Piwi-like proteins.
The unpaired last three bases of the strand of RNA
Structure and Function of Argonaute Proteins
1405

Figure 2. Proposed Exit Path for 3# End of RNA Targets

(A) Surface representation of the AfPiwi crystal structure colored by electrostatic potential.
(B) Ribbon diagram of the AfPiwi crystal structure oriented as in (A).
(C) Surface representation of the PfAgo crystal structure colored by electrostatic potential. (PfAgo, PDB code 1Z25.)
(D) Surface representation of a homology model of Drosophila melanogaster Ago2 based on the structure of PfAgo. This model was generated
by threading the sequence of DmAgo2 onto the PfAgo structure using SWISS-MODEL (http://www.expasy.org/spdbv/; Guex and Peitsch,
1997) and superimposing the crystal structure of the PAZ domain of DmAgo2 (Song et al., 2003) on the PAZ domain region.
For all panels: yellow, RNA strand representing the guide siRNA; pink, RNA strand representing the target RNA. For (A), (C), (D), and Figure
4B, electrostatic potential was calculated with GRASP (Nicholls et al., 1991) and is shown as blue for +10 kT/e and as red for −10 kT/e.

representing the target mRNA are visible in the crystal
structure from the Barford group (Parker et al., 2005)
and exit the protein through a channel between do-
mains A and B (middle and Piwi domains in full-length
Ago proteins). This cleft is basic and conserved in Af-
Piwi, PfAgo, and Ago2 (Figure 2). It is located on the
surface opposite that containing the proposed dsRNA
binding claw at the active site formed between the PAZ
and PIWI domains (Song et al., 2004).
The 5# phosphate group of the guide siRNA is bound
directly by side chains of four residues that are invariant
in Ago proteins (Y123, K127, Q137, and K163) and by
the main chain nitrogen of F138 (Figure 1). The phos-
phate group is also bound by a divalent cation, proba-
bly Mg2+, that is coordinated by Q159, the C-terminal
carboxylate, a water molecule, and the third phosphate
group of the guide RNA. This conserved basic pocket
along with a bound divalent cation was noted in the
structure of AfPiwi alone, and mutagenesis adding a
C-terminal extension interfered with siRNA binding
(Parker et al., 2004), suggesting that this could be the
5# phosphate binding pocket. Based on their crystal
structure, Ma et al. (2005) mutated Y123, K127, Q137,
and K163 to alanine, singly and in combination. These
mutations had modest effects on binding affinity (3- to
8-fold weaker binding), but an siRNA lacking the 5#
phosphate binds poorly to AfPiwi. To determine whether
this phosphate binding site is conserved in mammalian
Ago proteins, Ma et al. (2005) introduced the homolo-
gous mutations in human Ago2 and assessed the effect
on cleavage activity. The single alanine mutants K533A,
Q545A, and K570A were less active than wild-type
hAgo2 in cleavage assays and double mutants were se-
verely compromised. Thus, the binding of the 5# phos-
phate group observed in the AfPiwi structures appears
to represent a good model for the RNAi effector com-
plex. This 5# phosphate binding pocket is different from
that proposed based on soaking of PfAgo crystals with
tungstate, a phosphate group mimic (Rivas et al., 2005).
Assuming PfAgo binds to the guide siRNA as observed
for AfPiwi, the major tungstate binding site instead cor-
responds to the location of the phosphate group at the
Structure
1406
Figure 3. Superposition of the Active Sites of PfAgo (Blue) and Bh-
RNase HC (Green)
RNA strand from the DNA:RNA hybrid in the Bh-RNase HC struc-
ture is shown (pink). Water molecules from PfAgo (red) and Bh-
RNase HC (dark pink) are shown as spheres. Gray dashed lines,
coordination of metal A in PfAgo; red dashed lines, coordination of
metals A and B in Bh-RNase HC. (Bh-RNase HC, PDB code 1ZBI.)
3# end of the strand of RNA representing the mRNA
target in the AfPiwi structures.
After the unpaired first base of the guide strand, sev-
eral base pairs in A form conformation are visible (four
in the structure from the Patel group [Ma et al., 2005]
and five plus two unpaired bases in the structure from
the Barford group [Parker et al., 2005]). In both struc-
tures, the protein recognizes phosphate groups of the
second to fourth bases of the guide strand either di-
rectly or through a water-mediated contact. The path
of the RNA diverges slightly in the two structures after
the third base pair such that contacts are made to the
phosphate group of the fifth base in the Patel structure,
but not to the equivalent phosphate group in the Bar-
ford structure. No contacts are made to the additional
RNA backbone or bases in the Barford structure and,
in both structures, contact with the strand representing
the mRNA is minimal. This 5# bound complex corres-
ponds in part to the first state of a two-state model
for Ago mRNA recognition and cleavage proposed by
Tomari and Zamore (2005). In this model, the 5# end of
the guide siRNA binds to the target mRNA, while the 3#
end remains bound to the PAZ domain in the first state.
In the second state, the 3# end of the siRNA is released
from the PAZ domain to pair with the target RNA. This
fully paired second state has not yet been visualized.
Argonaute Active Site
The crystal structure of PfAgo reported about 1 year ago
revealed the similarity of the Piwi domain to RNase H,
and from this similarity two aspartic acids, D558 and
D628, had been identified as likely active site residues
(Song et al., 2004; Figure 3). Mutagenesis of the equiva-
lent residues in hAgo2 to alanine eliminated mRNA cleav-
age activity (Liu et al., 2004). But it had been more difficult
to predict a third metal-coordinating side chain, as this
residue is not well conserved spatially among RNase
H-like enzymes. Song et al. (2004) suggested E635 could
be a third metal-coordinating active site residue, and Yuan
et al. (2005) suggested recently that the equivalent resi-
due in AaAgo, E578, could be the third residue. Two gluta-
mate residues in hAgo2 could possibly align with E635 in
PfAgo or E578 in AaAgo. However, when mutants of these
two residues were created, E673A or E683G, mRNA
cleavage activity was not affected (Rivas et al., 2005).
To better define the active site of Ago proteins, Rivas
et al. (2005) soaked crystals of PfAgo in Mn2+, which can
replace the Mg2+ expected to be coordinated at the
active site but is easier to detect crystallographically. The
Mn2+ soaking experiment indicated that the third coordi-
nating side chain is H745 (Figure 3). In addition, the equiv-
alent residue in AaAgo, D683, was coordinated to a Ca2+
ion along with the two other conserved acidic residues in
the crystal structure of this protein (Yuan et al., 2005).
Mutation of the corresponding residue in hAgo2, H807,
also eliminated mRNA cleavage activity, confirming its im-
portance for catalytic activity (Rivas et al., 2005). Not all
Ago proteins are active as endonucleases, and the identi-
fication of the active site residues of this family of proteins
explains why some Ago proteins are not cleavage com-
petent. For example, in cleavage-incompetent hAgo1, the
catalytic histidine is replaced by arginine and mutation of
the equivalent histidine in cleavage-competent hAgo2 to
arginine inactivates it (Rivas et al., 2005). Similarly, in
cleavage-incompetent hAgo 4, one of the catalytic aspar-
tates is replaced by glycine.
Additional insight into the catalytic mechanism of
Ago proteins is provided by the recent crystal struc-
tures of a bacterial RNase H (Bh-RNase HC) with its
DNA:RNA hybrid substrate (Nowotny et al., 2005).
These structures provide a glimpse of how this family
of enzymes cleaves an RNA substrate. Nowotny et al.
(2005) found that the DNA:RNA hybrid binds at the
active site of BH-RNase HC via two divalent cations.
One is equivalent to the Mn2+ coordinated by two Asps
and a His in Ago proteins. This metal is also coordi-
nated by two water molecules, one that is positioned
w3 Å from the scissile phosphate group and is pre-
dicted to be the nucleophile (metal A; Figure 3). Equiva-
lent water molecules are found coordinating the Mn2+
ion in the PfAgo structure. A third water molecule is
coordinated to the Mn2+ and this corresponds to a non-
bridging oxygen atom in the phosphate group preced-
ing the scissile phosphate.
A second divalent cation (metal B; Figure 3) is coordi-
nated by an aspartate that coordinates both metals, a
glutamate, an asparagine, a water molecule, and the
RNA. It is more difficult to predict where this second
metal binds in the PfAgo structure. Two possible metal
ligands are E635 and R627 though they are more dis-
tant from the first metal ion than the ligands for the
second metal in the Bh-RNase HC structure (Nowotny
et al., 2005). The binding of the second metal ion likely
requires substrate binding, so it is not surprising that
Rivas et al. (2005) did not observe a second Mn2+ ion.
The two-metal ion catalytic mechanism likely is con-
served among RNase H family enzymes (Martinez and
Tuschl, 2004; Schwarz et al., 2004; Nowotny et al.,
2005), but identification of the ligands for the second
Structure and Function of Argonaute Proteins
1407
RNA; pink, target RNA. The arrow next to the PAZ domain indicates possible movement to accommodate the siRNA:target RNA duplex.
(B) Surface representation of the PfAgo crystal structure colored by electrostatic potential. Arrows indicate the locations of two possible
binding pockets for the 3# end of the siRNA fully paired to target RNA. The protein is oriented as in (A).
metal ion site may require a crystal structure of an Ago
protein with RNA bound at the active site.
Functions of Prokaryotic Argonaute Proteins
Prokaryotic Ago proteins have served as excellent mod-
els for learning about the structure and function of this
family of proteins. However, the function of these proteins
in their respective organisms was unknown when their
structures were determined. An alternative function was
hinted at in the paper by Ma et al. (2005) when they exam-
ined nucleic acid binding by AfPiwi protein. In equilibrium
binding assays, the AfPiwi protein bound more tightly to
ssDNA (Kd = 9 nM) than ssRNA (190 nM) and more tightly
to dsDNA (32 nM) than to a DNA:RNA hybrid (80 nM) or
dsRNA (190 nM). In the Patel group’s recent paper on the
structure of the AaAgo protein (Yuan et al., 2005), they
found that AaAgo also binds more tightly to ssDNA (10
nM) than to ssRNA (970 nM) and binds to a DNA:RNA
hybrid (640 nM) or dsDNA (1 ␮M), but does not seem to
bind to dsRNA. Having observed this, they examined the
ability of AaAgo preincubated with ssDNA or ssRNA to
direct cleavage of an RNA target. They found that AaAgo
could direct the cleavage of the RNA target when prein-
cubated with DNA or RNA. Cleavage directed by a DNA
guide was detectable when either Mn2+ or Mg2+ was
present, and cleavage with an RNA guide was detectable
only when Mn2+ was present. The most efficient cleavage
was directed by DNA oligonucleotides of 18, 21, and 24
nt in the presence of Mn2+ at 55°C. Other enzymes that
use Mg2+ as the physiological metal are more active
in vitro with Mn2+ (Pelletier et al., 1996; Cowan, 1998). The
location of the cleavage was at the same position as
when human Ago2 preincubated with ssRNA cleaved the
target RNA. These data suggest that AaAgo may function
as a DNA-directed ribonuclease in vivo.
Models of Argonaute Catalytic Complexes
Because the 5# end of the guide siRNA dictates the
location of the cleavage site, the crystal structures of
AfPiwi with the 5# end of the guide siRNA and paired
target mRNA along with the more accurately defined
active site from Rivas et al. (2005) provide a framework
for understanding how this might be accomplished.
Starting from the position of the 5# phosphate and fol-
lowing base pairs established in their respective struc-
tures, both the Barford and Patel groups modeled ex-
tended A form RNA from the base pairs observed in
Figure 4. Complexes of Ago Proteins and
Duplex RNA
(A) Ribbon diagram of the PfAgo crystal
structure with modeled RNA. This pro-
tein:RNA model was created by superimpos-
ing PfAgo on the crystal structure of AfPiwi
with a mimic of the 5# end of an siRNA paired
to a target RNA. The duplex RNA region was
extended to the 3# end of the siRNA by
lengthening the A form RNA (Parker et al.,
2005; AfPiwi and model RNA coordinates
courtesy of D. Barford). Blue, N-terminal do-
main; red, PAZ domain; green, middle do-
main; purple, Piwi domain; yellow, guide
their respective crystal structures of AfPiwi (Ma et al.,
2005; Parker et al., 2005). These exercises suggest that
the RNA when extended can place the scissile phos-
phate roughly at the active site.
If the full-length PfAgo or AaAgo structure is superim-
posed on the AfPiwi structure with extended model
RNA, it appears that the middle and Piwi domains may
need to move with respect to one another in order to
adjust the distance and place the scissile phosphate
more precisely at the active site (Figure 4A). Alterna-
tively, the RNA could form a bent or kinked conforma-
tion, but maintaining a straight A form conformation
seems more likely given the importance of base pairing
in this region of the siRNA:mRNA duplex. It also ap-
pears that the PAZ domain would need to move away
from the Piwi domain to open the clamp for the double-
stranded RNA (Figure 4A). This suggests that the PAZ
domain interacts with the guide siRNA strand, and this
interaction could help to correctly position the scissile
phosphate at the active site as well. The PAZ domains
in the PfAgo and AaAgo structures are oriented dif-
ferently with respect to the other three domains, sup-
porting the idea that alternate orientations are possible.
To produce a model of the AaAgo protein with a straight
DNA:RNA helix, Yuan et al. (2005) moved domains and
reoriented the trajectory of the DNA:RNA hybrid relative
to what was observed in the AfPiwi:RNA structure, sug-
gesting that Ago proteins may change conformations
upon RNA binding to correctly position the substrate.
From the active site toward the 3# end of the guide
RNA, a fully duplexed siRNA appears to have two pos-
sible paths, between the PAZ and N-terminal domains or
between the Piwi and N-terminal domains (Figure 4B). In
the model of an AaAgo DNA:RNA complex, the helical
molecule passes between the PAZ and N-terminal do-
mains (Yuan et al., 2005). However, by superimposing the
PfAgo or AaAgo structure on the Bh-RNase HC complex
with DNA:RNA hybrid (Nowotny et al., 2005), the hybrid
DNA takes a path from the active site that would fall be-
tween the Piwi and N-terminal domains. Thus, both path-
ways may be reasonable and await experimental confirm-
ation.
Conclusions
Thus far, the examination of crystal structures of Argo-
naute proteins has been instrumental in understanding
Structure
1408
their function, leading to the identification of it as the
elusive endonuclease of the RISC and explaining the
importance of siRNA/miRNA features, such as the 5#
phosphate group. Yet many outstanding questions leave
a field still ripe with opportunity to expand our under-
standing of this family of proteins. Continued structural
studies can guide us to an understanding of the forma-
tion of cleavage-competent complexes as well as those
that instead repress translation or transcription.
Acknowledgments
I am grateful to my colleagues at NIEHS for their critical comments,
Phillip Zamore for helpful discussions, and David Barford for shar-
ing the coordinates of his AfPiwi:RNA model.
Received: August 12, 2005
Revised: August 31, 2005
Accepted: August 31, 2005
Published: October 11, 2005
References
Brennecke, J., Stark, A., Russell, R.B., and Cohen, S.M. (2005).
Principles of microRNA-target recognition. PLoS Biol. 3, 404–418.
Cowan, J.A. (1998). Metal activation of enzymes in nucleic acid bio-
chemistry. Chem. Rev. 98, 1067–1088.
Doench, J.G., and Sharp, P.A. (2004). Specificity of microRNA target
selection in translational repression. Genes Dev. 18, 504–511.
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and
Tuschl, T. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA in-
terference in cultured mammalian cells. Nature 411, 494–498.
Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and
Tuschl, T. (2001b). Functional anatomy of siRNAs for mediating effi-
cient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20,
6877–6888.
Enright, A.J., John, B., Gaul, U., Tuschl, T., Sander, C., and Marks,
D.S. (2003). MicroRNA targets in Drosophila. Genome Biol. 5,
R1.1–R1.13.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and
Mello, C.C. (1998). Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391,
806–811.
Griffiths-Jones, S. (2004). The microRNA Registry. Nucleic Acids
Res. 32, D109–D111.
Guex, N., and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-
PdbViewer: an environment for comparative protein modeling.
Electrophoresis 18, 2714–2723.
Guo, S., and Kemphues, K.J. (1995). par-1, a gene required for es-
tablishing polarity in C. elegans embryos, encodes a putative Ser/
Thr kinase that is asymmetrically distributed. Cell 81, 611–620.
Haley, B., and Zamore, P.D. (2004). Kinetic analysis of the RNAi
enzyme complex. Nat. Struct. Mol. Biol. 11, 599–606.
Krek, A., Grun, D., Poy, M.N., Wolf, R., Rosenberg, L., Epstein, E.J.,
MacMenamin, P., da Piedade, I., Gunsalus, K.C., Stoffel, M., and
Rajewsky, N. (2005). Combinatorial microRNA target predictions.
Nat. Genet. 37, 495–500.
Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P., and
Burge, C.B. (2003). Prediction of mammalian microRNA targets.
Cell 115, 787–798.
Lewis, B.P., Burge, C.B., and Bartel, D.P. (2005). Conserved seed
pairing, often flanked by adenosines, indicates that thousands of
human genes are microRNA targets. Cell 120, 15–20.
Lim, L.P., Lau, N.C., Garrett-Engele, P., Grimson, A., Schelter, J.M.,
Castle, J., Bartel, D.P., Linsley, P.S., and Johnson, J.M. (2005).
Microarray analysis shows that some microRNAs downregulate
large numbers of target mRNAs. Nature 433, 769–773.
Lingel, A., Simon, B., Izaurralde, E., and Sattler, M. (2003). Structure
and nucleic-acid binding of the Drosophila Argonaute 2 PAZ do-
main. Nature 426, 465–469.
Lingel, A., Simon, B., Izaurralde, E., and Sattler, M. (2004). Nucleic
acid 3#-end recognition by the Argonaute2 PAZ domain. Nat.
Struct. Mol. Biol. 11, 576–577.
Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M.,
Song, J.J., Hammond, S.M., Joshua-Tor, L., and Hannon, G.J.
(2004). Argonaute2 is the catalytic engine of mammalian RNAi. Sci-
ence 305, 1437–1441.
Ma, J.B., Ye, K., and Patel, D.J. (2004). Structural basis for over-
hang-specific small interfering RNA recognition by the PAZ domain.
Nature 429, 318–322.
Ma, J.B., Yuan, Y.R., Meister, G., Pei, Y., Tuschl, T., and Patel, D.J.
(2005). Structural basis for 5#-end-specific recognition of guide
RNA by the A. fulgidus Piwi protein. Nature 434, 666–670.
Martinez, J., and Tuschl, T. (2004). RISC is a 5# phosphomonoester-
producing RNA endonuclease. Genes Dev. 18, 975–980.
Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and
Tuschl, T. (2002). Single-stranded antisense siRNAs guide target
RNA cleavage in RNAi. Cell 110, 563–574.
Matzke, M.A., and Matzke, A.J. (2004). Planting the seeds of a new
paradigm. PLoS Biol. 2, 582–586.
Nicholls, A., Sharp, K.A., and Honig, B. (1991). Protein folding and
association: insights from the interfacial and thermodynamic prop-
erties of hydrocarbons. Proteins 11, 281–296.
Nowotny, M., Gaidamakov, S.A., Crouch, R.J., and Yang, W. (2005).
Crystal structures of RNase H bound to an RNA/DNA hybrid: sub-
strate specificity and metal-dependent catalysis. Cell 121, 1005–1016.
Nykanen, A., Haley, B., and Zamore, P.D. (2001). ATP requirements
and small interfering RNA structure in the RNA interference path-
way. Cell 107, 309–321.
Parker, J.S., Roe, S.M., and Barford, D. (2004). Crystal structure of
a PIWI protein suggests mechanisms for siRNA recognition and
slicer activity. EMBO J. 23, 4727–4737.
Parker, J.S., Roe, S.M., and Barford, D. (2005). Structural insights
into mRNA recognition from a PIWI domain-siRNA guide complex.
Nature 434, 663–666.
Pelletier, H., Sawaya, M.R., Wolfle, W., Wilson, S.H., and Kraut, J.
(1996). A structural basis for metal ion mutagenicity and nucleotide
selectivity in human DNA polymerase β. Biochemistry 35, 12762–
12777.
Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon, G.J.,
and Joshua-Tor, L. (2005). Purified Argonaute2 and an siRNA form
recombinant human RISC. Nat. Struct. Mol. Biol. 12, 340–349.
Schwarz, D.S., Hutvagner, G., Haley, B., and Zamore, P.D. (2002).
Evidence that siRNAs function as guides, not primers, in the Dro-
sophila and human RNAi pathways. Mol. Cell 10, 537–548.
Schwarz, D.S., Tomari, Y., and Zamore, P.D. (2004). The RNA-
induced silencing complex is a Mg2+-dependent endonuclease.
Curr. Biol. 14, 787–791.
Song, J.J., Liu, J., Tolia, N.H., Schneiderman, J., Smith, S.K., Mar-
tienssen, R.A., Hannon, G.J., and Joshua-Tor, L. (2003). The crystal
structure of the Argonaute2 PAZ domain reveals an RNA binding
motif in RNAi effector complexes. Nat. Struct. Biol. 10, 1026–1032.
Song, J.J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L. (2004).
Crystal structure of Argonaute and its implications for RISC slicer
activity. Science 305, 1434–1437.
Stark, A., Brennecke, J., Russell, R.B., and Cohen, S.M. (2003). Identi-
fication of Drosophila microRNA targets. PLoS Biol. 1, 397–409.
Tomari, Y., and Zamore, P.D. (2005). Perspective: machines for
RNAi. Genes Dev. 19, 517–529.
Yan, K.S., Yan, S., Farooq, A., Han, A., Zeng, L., and Zhou, M.M.
(2003). Structure and conserved RNA binding of the PAZ domain.
Nature 426, 468–474.
Yuan, Y.R., Pei, Y., Ma, J.B., Kuryavyi, V., Zhadina, M., Meister, G.,
Chen, H.Y., Dauter, Z., Tuschl, T., and Patel, D.J. (2005). Crystal
structure of A. aeolicus Argonaute, a site-specific DNA-guided en-
doribonuclease, provides insights into RISC-mediated mRNA
cleavage. Mol. Cell 19, 405–419.