REVIEW

Slicer and the Argonautes

© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

Niraj H Tolia & Leemor Joshua-Tor

Though they started out as somewhat mysterious components of the RNAi effector complexes, Argonaute proteins have since
taken center stage in RNAi gene silencing. They interact with small RNAs to effect gene silencing in all RNAi-related pathways
known so far. We will review the dramatic advances in our understanding of the role of the Argonautes in RNAi through studies of
their structure and function.

RNA interference (RNAi) has emerged as a new means for controlling
gene expression at the transcriptional, post-transcriptional and trans-
lational levels. Owing to its apparent overarching control and ease of
manipulation, RNAi has been exploited as a tool for investigating gene
function through silencing. Several recent discoveries have elucidated
through slicing. During translational repression, RISC mediates silencing
of mRNA transcripts without target slicing by binding and sequestering
transcripts away from the translational machinery into cytoplasmic foci
a b

Drosha
Dicer
some of the molecular players and their roles in the mechanism of 3'
5' Nucleus
RNAi, in particular those involved in transcriptional and post-transcrip- TRBP/R2D2 DGCR8/Pasha
siRNA
tional gene silencing (TGS and PTGS, respectively). In this review, we pri-miRNA
focus on the protein that is at the heart of all RNAi effector complexes: Ago pre-miRNA
Argonaute. Cytoplasm
3' 5' Dicer

Effector complexes of RNAi TRBP/R2D2

miRNA
During RNAi, long double-stranded RNA is processed to yield small Passenger strand 10 nt
(~19–31 nucleotide) double-stranded RNAs (Box 1 and Table 1) that cleavage
Ago
trigger the RNAi response. One strand of a small RNA (the guide strand,
3' 5'
which is antisense to the target RNA) is incorporated into the effector
complexes of RNAi, which are called RNA-induced silencing complexes
(RISCs)1 (Fig. 1). The minimal defining features of RISC include the 3' 5'
Bypass
mechanism Helicase?
presence of an Argonaute family member and the guide strand of a small
RNA. RISC mediates several different modes of silencing.
One method of post-transcriptional silencing is sequence-specific 3' 5'
Slicing
degradation of mRNA targets, which is called ‘slicing’ (Fig. 1a). This cycle
80S
is the pathway most often used for RNA gene-knockdown technology 10 nt
and by small interfering RNAs (siRNAs). During slicing, the guide RNA 30S

that has been incorporated into RISC directs binding of the complex
to its cognate target RNA through base complementarity. RISC then
cleaves the target RNA at the position opposite the phosphate between
the tenth and eleventh base from the 5′ end of the guide. This cleav-
age event results in silencing of the target mRNA by preventing read-
through of the message by the translational machinery and leads to
mRNA destruction.
An alternate post-transcriptional outcome is translational repression
(Fig. 1b). This method of silencing is most often used by microRNAs
as a means for endogenous gene regulation. However, plant and cer-
tain mammalian microRNAs2 can also facilitate message degradation
W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory,
1 Bungtown Road, Cold Spring Harbor, New York 11724, USA. Correspondence
should be addressed to L.J. (leemor@cshl.edu).
Published online 15 December 2006; doi:10.1038/nchembio848
P-bodies
Figure 1 Pathways of post-transcriptional gene silencing. (a) Slicing cycle
(also called RNAi) via siRNAs. Dicer processing of long double-stranded
RNA results in the formation of siRNA, which is then loaded onto Argonaute
(Ago). One strand is cleaved (passenger strand cleavage) and Ago is primed
for target mRNA degradation. The Ago–guide RNA complex then binds
and cleaves the target RNA. Finally, the complex is turned over, releasing
the cleaved RNA, and is able to recognize and cleave additional substrate
molecules. (b) Translational suppression via microRNAs (miRNAs). Drosha
processing of pri-miRNA results in pre-miRNA that is exported from the
nucleus. Pre-miRNA is substrate for Dicer, which produces the mature
miRNA that is loaded onto Ago. One strand of the miRNA is removed by
the bypass mechanism, thereby allowing Ago to bind to target mRNA,
which inhibits translation. Translation repression is thought to occur by the
trafficking of targeted transcripts to cytoplasmic foci called P-bodies that are
devoid of the translation machinery. nt, nucleotide.

36 VOLUME 3 NUMBER 1 JANUARY 2007 NATURE CHEMICAL BIOLOGY

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BOX 1 SMALL RNAS

Several types of small RNAs (sRNAs) have been identified (Table 1). Biogenesis of these small RNAs is for the most part dependent on the
RNase III-type enzyme Dicer. Dicer processing results in small RNA products harboring 5′ phosphates and 3′ two-nucleotide overhangs on
each strand. siRNAs, miRNAs, tasiRNAs, tncRNAs and scnRNAs are all dependent on Dicer for biogenesis. However, two classes of small
RNAs, rasiRNAs and piRNAs, seem to be formed independently of Dicer processing.

Table 1 Classes of small RNAs

Class Description Size (nt) Observed in Mode of action Biogenesis
siRNA Small interfering RNA ~20–24 Mammals, Tend to have perfect complementarity to their mRNA Dicer processing of long dsRNA81
At, Dm, Sp, Ce target. Can be found dispersed throughout the entire (Fig. 1a)
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

message. Result in mRNA degradation

miRNA MicroRNA ~20–24 Mammals, Mammalian miRNAs tend to contain mismatches, usually Two-step processing of primary
At, Dm, Sp, Ce target untranslated regions of target mRNAs and result in miRNA transcripts (pri-miRNA), first
translational suppression. Certain mammalian miRNAs by the microprocessor complex82,83
can also facilitate mRNA degradation. Plant miRNAs tend and then by Dicer84–86 (Fig. 1b)
to have perfect complementarity to their target and
facilitate message degradation
tasiRNA trans-acting siRNA ~24 At, Dm, Sp Act like siRNAs facilitating mRNA degradation87. Dicer processing of RdRP-derived
However, tasiRNAs act in trans, targeting transcripts of long dsRNA87
genes other than the gene they are derived from
tncRNA Tiny noncoding RNA ~20–21 Ce Like tasiRNAs, tncRNAs also act in trans, resulting in tncRNAs are derived from noncoding
message degradation88,89. May also facilitate sequences via Dicer processing in
translational suppression the nematode worm88,89
scnRNA Small scan RNA ~28 Tt Function in DNA elimination Dicer-dependent small RNAs90–92
rasiRNA Repeat-associated ~24–29 At, Dm, Sp, Ce Result in transcriptional silencing via chromatin remodel- Biogenesis is unclear, but rasiRNAs
small interfering RNA ing (see Slicing and TGS) match repetitive sequence
elements13–22
piRNA Piwi-interacting RNA ~26–31 Mammals Specifically expressed in the germ line, where they Biogenesis unclear, likely through
function in gametogenesis53–57. Mode of action is unclear processing of transcripts
At, A. thaliana; Dm, D. melanogaster; Sp, S. pombe; Ce, C. elegans; Tt, T. thermophila; RdRP, RNA-dependent RNA polymerase; dsRNA, double-stranded RNA.

termed P-bodies3–6. The exact mechanism of translational repression is
only beginning to be unraveled and is an area of intense study. Efficient
translational repression is thought to require binding of RISC to mul-
tiple target sequences within a given mRNA (Fig. 1b).
A third method of silencing is TGS, which was initially observed in
plants7. TGS has also been demonstrated in Schizosaccharomyces pombe8
and Drosophila melanogaster9. In S. pombe a specialized form of RISC,
called the RNA-induced initiation of transcriptional silencing (RITS)
complex, facilitates small RNA–mediated TGS by regulation of hetero-
chromatin.
RISC was originally identified through fractionation of sequence-
specific nuclease activity from D. melanogaster extracts10. Subsequent
protein sequencing of active fractions revealed several components,
including Argonaute 2 (DmAgo2), Vasa intronic gene (VIG), the
D. melanogaster homolog of fragile X protein (DmFXR) and Tudor-SN11–13.
Of these proteins, Argonaute was of particular interest as it was always
present in the various purifications of RISC from diverse species, includ-
ing the RITS complex. Argonautes contain two signature domains, PAZ
and PIWI. The PAZ domain is also found in Dicers, the enzymes respon-
sible for production of small RNAs from long double-stranded RNA.
Thus, these domains seem to be exclusive to RNAi-related proteins. The
crystal structure of Argonaute from Pyrococcus furiosus (PfAgo; Fig. 2)
revealed an RNase H-like PIWI domain, and together with subsequent
structure-based mutagenesis of HsAgo2 it established Argonautes as the
catalytic core of the effector complex, RISC14,15. The RNase H-like fold
was later also shown by the structure of the Archaeoglobus fulgidus PIWI
protein (AfPIWI)16 and by the structure of Aquifex aeolicus Argonaute
(AaAgo)17. RNase H enzymes are Mg2+-dependent RNases that cleave
DNA-RNA hybrids, which results in a 5′ product carrying a 3′ hydroxyl
and a 3′ product carrying a 5′ phosphate18. Although RISC is an RNA-
guided RNase, RISC cleavage (slicing) results in products having ends
similar to those of products of RNase H-type enzymes19,20 and is also
dependent on divalent cations20.
Of the human Argonautes 1–4, only RISC-containing human
Argonaute 2 (HsAgo2) is cleavage-competent15,21. Different reports
suggested that the molecular weight of RISC is between 140 and 500
kDa10,22,23. It was later demonstrated that bacterially expressed and puri-
fied HsAgo2 and a small RNA are the only elements required to recon-
stitute this slicing activity24. This result is in agreement with the lowest
molecular weight estimate for RISC given above, and further suggests
that the wide range of molecular weights reported for RISC represent
several different versions of the complex that contain other factors in
addition to Argonaute. Because the other components of RISC are not
required for slicing, they may have a role in other aspects of RISC activ-
ity, for example substrate turnover and/or RISC subcellular localization.
Furthermore, fractionation of RISC activity followed by mass spectro-
metric analysis revealed that HsAgo2 was the only remaining protein25.
Finally, mutation of catalytic residues in HsAgo2, which were identified
from the crystal structure of PfAgo, abolishes RISC activity15,24. Together
these results demonstrate that HsAgo2 is Slicer, the enzyme in RISC that
is responsible for mRNA cleavage.
Argonaute structure and function
Argonautes are composed of four domains: the N-terminal, PAZ, mid-
dle and PIWI domains (for recent review see ref. 26). The first crys-
tal and NMR structures to be solved were of PAZ domains from fly
Argonautes27–29. These structures revealed that the PAZ domain has a
deviant OB fold, which suggests a role in nucleic acid binding. The PAZ
domain does indeed bind RNA, as shown by biochemical methods27–29
and by structures of PAZ in complex with nucleic acids30,31. Binding is
in a sequence-independent manner, with the PAZ domain recognizing
the single-stranded 3′ end of siRNA.

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 1 JANUARY 2007 37

 REVIEW
The structure of full-length Argonaute from PfAgo (Fig. 2), which was
solved subsequently, was instrumental in defining roles for the remain-
ing domains and revealing the function of Argonaute14. As mentioned
above, the PIWI domain has an RNase H fold. Strikingly, in PfAgo, in
addition to conserved secondary structure elements of the fold, two
aspartates that are critical active site residues in other RNase H fam-
ily proteins and are always found in identical positions on adjacent
β-strands are also conserved. A more detailed discussion of the active
site follows. The middle domain has structural homology to the sugar-
binding domain of lac repressor. In lac repressor, two such sugar-bind-
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
ing domains sandwich a single lactose molecule. The crystal structure
of AfPIWI16, which has both middle and PIWI domains but lacks the
N-terminal and PAZ domains, followed by the crystal structure of full-
length AaAgo17, also demonstrated the similarity of the middle and
PIWI domains to lac repressor and RNase H, respectively.
In PfAgo, the N-terminal, middle and PIWI domains form a crescent-
shaped base, and the PAZ domain is held above this base via a “stalk”
region14,17. This organization creates positively charged grooves that
may be nucleic acid binding sites. The largest groove is formed between
the PAZ and the crescent base, and a smaller groove is found between
the N-terminal and PIWI domains. Modeling of siRNA and target bind-
ing starting from the PAZ 3′ end binding site has revealed that the large
groove can accommodate the duplex positioning the scissile phosphate
at the active site.
Two-metal ion catalysis of RNase H proteins
RNase H domains are found in other well-characterized proteins.
Retroviral integrases and transposases are RNase H-like enzymes that
catalyze two consecutive reactions, donor-end processing and nucleo-
tidyl transfer, resulting in strand transfer32. Escherichia coli RNase H1
catalyzes a single reaction resulting in substrate cleavage. For cleavage
and donor-end processing reactions, the nucleophile is a water molecule,
and in nucleotidyl transfer reactions the nucleophile is a nucleotide
Asp558
Asp628
His745
Figure 2 The crystal structure of Argonaute from P. furiosus. The N-terminal
domain is in blue, the PAZ domain is red, the middle domain is green and
the PIWI domain is purple. The PAZ domain is held above the crescent-
shaped base created by the N-terminal, middle and PIWI domains. A closeup
of the active site residues (Asp-Asp-His) coordinating an Mn2+ ion is also
shown. Mn2+ was used in crystallization to characterize the active site, as it
easier to distinguish crystallographically and can functionally replace Mg2+,
the natural ion that is coordinated by the Ago Asp-Asp-His motif.
38
3′-OH. In all cases, the nucleophile is activated by a metal ion. Reactions
occur in a one-step nucleophilic substitution (SN2)-like mechanism and
proceed via a pentacovalent intermediate leading to inversion of the
phosphate stereoconfiguration33,34. This scheme follows a two-metal-
ion catalysis mechanism, with one metal activating the nucleophile and
the second stabilizing the intermediate35,36. To perform these reactions,
RNase H proteins use a conserved Asp-Asp-Glu/Asp motif for diva-
lent metal ion binding and catalysis36. Two Mg2+ ions are observed in
substrate-bound complexes of Tn5 transposase37 and Bacillus halodu-
rans RNase H1 structures36, coordinated by an Asp-Asp-Glu motif. In
the RNase H1 structure, an additional aspartate and the nucleic acid
substrate also coordinate the metal ions. As described above, RNase H
cleavage, like RISC slicing, creates a 5′ product with a 3′ hydroxyl and a
3′ product carrying a 5′ phosphate.
Slicer activity
A combination of crystallographic and mutation analyses determined
that, although the two critical aspartates are present, Argonautes use an
additional histidine rather than another carboxylate residue for catalysis
(Fig. 2)24. Thus, in contrast to other RNase H proteins, Argonautes use
a conserved Asp-Asp-His motif. In manganese-soaked PfAgo crystals,
only one metal ion was observed to be coordinated by the Asp-Asp-His
motif. This one-metal ion binding is analogous to the substrate-free
structures of RNase H1 and Tn5 transposase, as the second metal ion
might require the presence of substrate for coordination. Other than the
difference in metal-binding residues, the catalytic mechanism of Slicer
is not thought to be very different from the two-metal-ion mechanism
proposed for canonical RNase H proteins.
Of the eight human Argonaute family members, only HsAgo1, HsAgo2,
HsAgo3 and HsAgo4 have been tested for activity, with HsAgo2 alone
having Slicer activity15,21. Arabidopsis thaliana has ten Argonaute family
members, two of which have been tested for and possess demonstrable
Slicer activity (AtAgo1 and AtAgo4)38–40. Of the four D. melanogaster
Argonautes, DmAgo141, DmAgo225 and DmPiwi42 have been tested for
and all possess Slicer activity. The single S. pombe Argonaute (SpAgo1)
is an active Slicer43, which is consistent with the demonstration of post-
transcriptional silencing in this organism44. Caenorhabditis elegans has
the largest known number of Argonaute family members: 24. Although
some of the worm Argonautes have been genetically linked to PTGS (for
example CeRde-145), and although they are presumed to function via slic-
ing, at present none of these have been tested in vitro for Slicer activity.
As mentioned above, DmPiwi has been shown to possess Slicer activ-
ity even though it contains a lysine instead of a histidine, which results
in an Asp-Asp-Lys active site42 (Table 2). It is also plausible that carbox-
ylates could functionally replace histidine, as aspartates or glutamates
are observed at this position in other RNase H proteins. Together these
results suggest that the Slicer catalytic motif is moderately degenerate
and should be defined as Asp-Asp-Asp/Glu/His/Lys.
The conservation of the Asp-Asp-His motif partially explains the
activity of the different Argonautes (Table 2). In humans, HsAgo1 has
an arginine instead of the histidine, and in HsAgo4 the second aspartate
is a glycine, perhaps explaining why both Argonautes are inactive when
immunoprecipitated from human cells. However, both HsAgo2 and
HsAgo3 have complete Asp-Asp-His motifs, yet only immunoprecipi-
tated HsAgo2 is active15,21. Why this is the case is still unclear.
For the remainder of our discussion, Argonaute family members that
have demonstrable slicing activity are termed Slicers, those that have an
intact catalytic motif are termed Slicer-like and those that do not contain
an intact motif are termed non-Slicers.
The presence of an Asp-Asp-His motif is required but is not suffi-
cient for Slicer activity (HsAgo3 being a case in point). As described
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Table 2 Catalytic residues of Argonautes

HsAgo1 VIFLGA D V IFYR D GVP IPAPAYYA R LVAF Ago
HsAgo2 VIFLGA D V IFYR D GVS IPAPAYYA H LVAF Ago
HsAgo3 VIFLGA D V IFYR D GVS IPAPAYYA H LVAF Ago
HsAgo4 VIFLGA D V IYYR G GVS IPAPAYYA R LVAF Ago
HsHIWI/PIWIL1 VMIVGI D C IVYR D GVG VPAPCQYA H KLAF Piwi
HsHILI/PIWIL2 LMVIGM D V VVYR D GVS VPAPCKYA H KLAF Piwi
HsPIWIL3 TMFVGI D C IVYR D GVG VPAPCHYA H KLAY Piwi
HsHIWI2/PIWIL4 LMVVGI D V IVYR A GVG VPAPCQYA H KLTF Piwi
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

AtAgo1 TIIFGA D V IFYR D GVS IVPPAYYA H LAAF Ago

AtAgo2 VMFIGA D V VIFR D GVS LVPPVYYA D MVAF Ago
AtAgo3 VMFIGA D V VIFR D GVS LVPPVSYA D KAAS Ago
AtAgo4 TIILGM D V IIFR D GVS VVAPICYA H LAAA Ago
AtAgo5 TIIMGA D V IFYR D GVS IVPPAYYA H LAAF Ago
AtAgo6 TLILGM D V IIFR D GVS SVAPVRYA H LAAA Ago
AtAgo7 VIFMGA D V IFFR D GVS IVPPAYYA H LAAY Ago
AtAgo8 TIIIGM D V IFYR D GVS VVAPVCYA H LAAA Ago
AtAgo9 TIIVGM D V IIFR D GVS VVAPVCYA H LAAA Ago
AtZll/Pnh TIIFGA D V IFYR D GVS IVPPAYYA H LAAF Ago

DmAgo1 VIFLGA D V ILYR D GVS IPAPAYYA H LVAF Ago

DmAgo2 TMYIGA D V IYYR D GVS YPAPAYLA H LVAA Ago
DmAUB LMTVGF D V LFFR D GVG VPAVCHYA H KLAF Piwi
DmPIWI LMTIGF D I VFYR D GVS VPAVCQYA K KLAT Piwi

SpAgo1 TLILGG D V IYFR D GTS LVPPVYYA H LVSN Ago

CeZK757.3 TMVVGI D V IVYR D GVS IPTPVYYA D LVAT Ago

CeT22B3.2 TMVVGI D V IVYR D GVS IPTPVYYA D LVAT Ago
CeT23D8.7 VLFIGC H L IIYR A GIA IPSPVYYA K LVAQ Ago
CeAlg-1 VIFFGC D I VVYR D GVS IPAPAYYA H LVAF Ago
CeAlg-2 VIFLGC D I VVYR D GVS IPAPAYYA H LVAF Ago
CeR09A1.1/CeERGO-1 TLVLGI D V VVYR D GLS LPAPVLYA H LAAK Piwi
CePRG-1 TMIVGY D L ILYR D GAG VPAPCQYA H KLAF Piwi
CePRG-2 TMIVGY D L ILYR D GAG VPAPCQYA H KLAF Piwi
CeRde-1 TMYVGI D V VVYR D GVS LPVPVHYA H LSCE Group 3
CeF20D12.1/CeCSR-1 TFVIGM D V IIFR D GVS IPTPVYVA H ELAK Group 3
CeC04F12.1 TLIISY D V VILR D GVS LPESIYAA D EYAK Group 3
CeM03D4.6 LLLIGL S T VIYL C GMS LPAPLYLT A EMAE Group 3
CeK12B6.1/CeSAM-1 RLIIGF E T LIYF S GVS LPIPLHIA G TYSE Group 3
CeF56A6.1/CeSAM-2 RLIVGF V T LLYF N GVS LPVPLYIA D RYSQ Group 3
CePpw-1 RLIVGF V T LLYF N GVS LPVPLYIA D RYSQ Group 3
CeZK1248.7 QLIIGV G V IIYR S GAS IPTPLYVA N EYAK Group 3
CeF58G1.1 HLIIGV G I IVYR T GTS LPTPLYVA N EYAK Group 3

CeC06A1.4 HLIIGV G I IIYR T ETS LPTPLYVA N EYAK Group 3

CePpw-2 HLIIGV G I TIYR S GSS IPTPLYVA N EYAK Group 3
CeF55A12.1 QLIIGV G V IIYR S GAS IPTPLYVA N EYAK Group 3
CeR06C7.1 QLIIGV G V IIYR S GAS IPTPLYVA N EYAK Group 3
CeY49F6A.1 TQFIGF E M VIYR T GAG VPHILYAA D NLAK Group 3
CeR04A9.2 TQFIGF E M VVYR V GSG IPNVSYAA Q NLAK Group 3
CeT22H9.3 VQFIGF D I VIYR I GAG FPDVLYAA E NLAK Group 3
CeC16C10.3 VQFIGF E I VIYR V GAG VPDVLYAA E NLAK Group 3
CeC14B1.7 VQFIGF E I VIYR V GAG VPDVLYAA E NLAK Group 3
Argonautes shown are from H. sapiens (Hs), A. thaliana (At), D. melanogaster (Dm), S. pombe (Sp) and C. elegans (Ce). Critical aspartates are colored in red, and
histidines are colored in blue. Substitutions for the Asp-Asp-His motif, such as aspartate to glutamate or histidine to either aspartate or glutamate, are colored in light
red. Substitution of the histidine to a lysine is colored in light blue.

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 1 JANUARY 2007 39

 REVIEW

above for HsAgo1 and HsAgo4, HsHIWI2

does not have a complete Asp-Asp-His motif HsAgo4
HsAgo1

CeF56A6.1/CeSAM-2
(Table 2), making it a non-Slicer, and it is Group 3

-1
CePpw-1

SR
Ce 12.1 CeC
unlikely to have Slicer activity. Notably, in CeK12B6.1/CeSAM-1

/
CeF58G1.1

4F 2.1
HsAgo2

.7
48
912 CePPW-2

C0 D1
A. thaliana all ten Argonaute family members

12
Ce F20
CeM03D4.6

ZK
1,000

Ce
HsAgo3 CeC06A1.4
have either a complete Asp-Asp-His or com- 629
1,000 1,000
plete Asp-Asp-Asp motif, which suggests that CeAlg-1 891
997
992

all are Slicer-like and could be active Slicers39. 1,000 1,000 699
682 1,000 1,000 CeR06C7.1
833 1,000
The only fly Argonaute not to have been tested CeAlg-2
CeRde-1 503

992 700 CeF55A12.1

CeY49F6A.1
for Slicer activity (DmAub) has a complete DmAgo1
CeT23D8.7
981
CeR04A9.2
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

291 368
motif, which suggests that, as in A. thaliana, CeZK757.3 398
999
CeT22H9.3
972 666
all fly Argonautes are active Slicers. The sole CeT22B3.2
729 1,000 CeR09A1.1/
CeERGO-1
943
CeC16C10.3
Argonaute of S. pombe (SpAgo1) does have a Ago-like AtAgo3
1,000
464 612
840
834

DmA
1,000
complete catalytic motif (Asp-Asp-His) and, as 956 1,000

go2
1,000 512 972
AtAgo2 HsHILI
described above, is an active Slicer. Of the 24

SpA
AtAgo7 1,000 CeC14B1.7
1,000

go1
C. elegans Argonautes only ten have an intact 1,000
718
HsHIWI2

DmPIWI
CePRG-2
catalytic motif, which suggests a great deal of AtZll/Pnh 1,000

CePRG-1
DmAUB
variation in both Slicer-like and non-Slicer HsHIWI

At
AtAgo1 AtAgo5

Ag
913

o6
HsPIWIL3
Argonautes in the worm. AtAgo4
Piwi-like
AtAgo8
Breaking down the Argonautes AtAgo9

A previous phylogenic analysis of several

Argonautes suggested that they can be divided
Figure 3 The three clades of the Argonautes. Multiple sequence alignment was performed using
into the Ago-like and Piwi-like subfamilies,
ClustalW (version 1.83; http://www.ebi.ac.uk/clustalw). Phylip (version 3.65; http://evolution.genetics.
based on similarity to AtAgo1 and DmPiwi, washington.edu/phylip.html) was used for bootstrap analysis, protein distance calculation (using
respectively46. The Piwi-like proteins contain Dayhoff PAM distance) and tree construction. Bootstrap values are indicated at each fork. Argonautes
four domains, like Argonautes, and are not that contain a complete catalytic motif and/or were shown to have Slicer activity are underlined.
to be confused with the two-domain protein
AfPIWI. Some of the Piwi-like Argonautes
have a role in stem cell self-renewal47–49. DmPiwi and DmAub (the fly and Piwi-like clades contain a high proportion of proteins from all spe-
Piwi-like Argonautes) are expressed in germ cells of the fly, and muta- cies that are either Slicers or Slicer-like. Thus, the worm has evolved
tion of either results in abnormal development of sperm cells47,50. a large set of non-Slicer Argonautes. Inclusion of 21 Argonautes from
Similarly, the mouse Piwi-like Argonautes, MmMILI and MmMIWI, C. briggsae in a similar analysis showed that C. briggsae also has group 3
are expressed in sperm cell precursors; knockout of either results in Argonautes. Therefore, the divergence of the group 3 Argonautes arose
defects in spermatogenesis51,52. before the C. elegans–C. briggsae split. CeRde-1 clusters in this clade.
Recently, examination of the total small RNA populations in the testes CeRde-1 was the first Argonaute family member to be genetically linked
of mammals revealed an abundance of small RNAs 26–31 nucleotides to silencing45, as CeRde-1 mutants have a penetrant loss of RNAi. Two
in length53–57. These small RNAs seemed to be exclusively bound by the additional members of this clade, CePpw-1 and CePpw-2, seem to be
Piwi-like Argonautes. Thus, this class of small RNAs was termed Piwi- involved in RNAi in the germ line of C. elegans59,60.
interacting RNAs (piRNAs). In addition to their longer length, piRNAs Extensive genetic analysis of the Argonautes in C. elegans has revealed
are unusual in that they have a preference for a uracil at their 5′ end. that specific Argonautes act sequentially and has also defined roles for
piRNAs are found clustered in short genomic loci, with the piRNAs several Argonautes, including those in the novel clade of worm-specific
within each cluster being generated in the same orientation, suggesting Argonautes58. A loss of both germ-line and somatic RNAi in the worm
a strand bias during biogenesis. The strand bias further suggests that mul- has only been achieved by a combined mutation of six Argonautes:
tiple piRNAs may be processed from a single long transcript. However, CePpw-1, CeK12B6.1/CeSam-1, CeF58G1.1/CeSam-2, CeF58G1.1,
piRNA precursors are not predicted to fold into double-stranded stem- CeC06A1.4 and CeM03D4.6. All of these are found in the novel clade of
loop structures, and the exact method of piRNA biogenesis is still unclear. group 3 Argonautes. The authors of this study also showed that during
Notably, the piRNA complex from rat testes, which contains Riwi (a RNAi, CeRde-1 binds to primary siRNA produced from Dicer process-
Piwi-like Argonaute), has demonstrable Slicer activity56. Riwi does have ing of exogenous double-stranded RNA. This CeRde-1–siRNA complex
a catalytic Asp-Asp-His motif, consistent with it being an active Slicer. then targets mRNA and recruits the RNA-dependent RNA polymerase,
As described above, DmPiwi also has demonstrable Slicer activity. These thereby resulting in amplification of the target mRNA. This product is
results raise the possibility that additional Piwi-like Argonautes may also then a substrate for Dicer, which leads to secondary siRNA production.
be active Slicers. However, piRNAs do not seem to be complementary to The secondary siRNAs are incorporated into the group 3 Argonautes,
mRNAs, which suggests that they do not regulate protein translation. which then mediate further silencing of the target. Given that most of
Thus, the modes of action of piRNAs and the Piwi-like Argonautes (and the group 3 Argonautes are non-Slicer-like, the secondary silencing is
the role of their Slicer activity) still need clarification. probably mediated by translational repression.
Another phylogenic analysis of Argonautes from Homo sapiens,
A. thaliana, C. elegans, Caenorhabditis briggsae, D. melanogaster and Argonaute RNA binding
S. pombe suggests the existence of a third clade that we have termed As mentioned earlier, the PAZ domain binds the 3′ end of the guide
the group 3 Argonautes58 (Fig. 3). This clade is worm-specific and pre- RNA. Recently, two crystal structures of AfPIWI in complex with siRNA-
dominantly contains non-Slicer Argonautes. In contrast, the Ago-like like duplexes have been solved61,62. These structures suggest a mode of

40 VOLUME 3 NUMBER 1 JANUARY 2007 NATURE CHEMICAL BIOLOGY


recognition of the 5′ end of the siRNA. The 5′ nucleotide of the siRNA
is unpaired, and the base and phosphate interact with residues of the
middle domain via base-stacking and ionic interactions, respectively. This
is consistent with the observation that nucleotides 2–8 of the guide RNA
(the ‘seed’ sequence) have a large role in target recognition and specific-
ity63–65. Coordination of the 5′ phosphate is also dependent on a metal
ion that is in turn bound to the C terminus of the protein. This metal ion
is different from the one coordinated by the catalytic residues. However,
the metal ion is not observed in the apo and manganese-soaked crystals
of PfAgo and may require the presence of the guide strand for coordina-
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
tion if the mode of siRNA binding is similar between these proteins. The
remainder of the duplex exits the protein via a basic surface formed by
the middle and PIWI domains. Both this basic surface and several of the
5′ interaction residues are conserved in PfAgo and human Argonautes.
A separate position for the 5′ binding site, located near the PIWI box in
the PIWI domain, has been proposed based on soaking of PfAgo crystals
with the phosphate mimic tungstate24. A possible model of PfAgo duplex
binding has been proposed based on a study in which the investigators
overlaid the AfPIWI–RNA duplex structure over the middle and PIWI
domains and extended the RNA duplex66. This model suggests that the
tungstate binding site might represent a phosphate binding site from the
3′ end of the target RNA. However, the correct mode of binding awaits
experimental confirmation.
Guide RNA loading and substrate specificity
Argonaute substrate specificity is determined by the sequence of the
bound guide RNA, as the target binds via hybridization to the guide
strand. One strand of the double-stranded siRNA or microRNA is incor-
porated into RISC (Fig. 1). This process is mediated by Dicer, human
immunodficiency virus-1 transactivating response (TAR) RNA-bind-
ing protein (TRBP; or its fly homolog R2D2) and Argonaute, and is
dependent on thermodynamic differences between the two ends of the
double-stranded siRNA (for recent review see ref. 67). The strand con-
taining the 5′ end with lower thermodynamic stability of base pairing is
incorporated into mature RISC (the guide strand), and the other strand
(the passenger strand) is destroyed.
Several different complexes have been proposed for the process of
loading RISC with guide RNA (reviewed in ref. 67). One of these is the
RISC loading complex (RLC), which (in the fly) loads DmAgo2 and is
a multiprotein complex that includes Dicer and R2D2 amongst other
components. The RLC binds to both ends of double-stranded siRNA;
R2D2 is the asymmetry sensor that recognizes the 5′ end with higher
thermodynamic stability of base pairing20. Strand separation and RISC
loading has been proposed to then occur either by (i) transfer of the
duplex to Argonaute followed by Argonaute-mediated passenger strand
cleavage (the “passenger strand cleavage mechanism;” Fig. 1a)41,68–70
or (ii) RNA unwinding of the double-stranded siRNA via an as-yet-
unidentified helicase followed by transfer of single-stranded guide RNA
to Argonaute (the “bypass mechanism;” Fig. 1b).
Passenger strand cleavage requires an active slicing Argonaute and as
such cannot be invoked for inactive Argonautes such as HsAgo1, HsAgo3
and HsAgo4, which presumably are loaded by the bypass mechanism.
Furthermore, recombinant HsAgo2 (rHsAgo2) is active for slicing tar-
get RNA when primed with single-stranded guide RNA but not double-
stranded guide RNA24, which suggests that rHsAgo2 either cannot be
loaded by double-stranded guide RNA or is unable to unwind the two
strands by itself.
In addition to thermodynamic asymmetry, guide RNA loading is
also dependent on the presence of a 5′ phosphate for ‘licensing’, or entry
into the RLC23. The 5′ phosphate also enhances binding of the guide
to Argonaute and is important for RISC cleavage fidelity24. An siRNA
NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 1 JANUARY 2007
REVIEW
with a 5′ phosphate forms more recombinant RISC and can exchange
for nonphosphorylated siRNA in competition assays, which suggests that
Argonaute has a higher affinity for phosphorylated siRNA. Furthermore,
cleavage with nonphosphorylated siRNA could be shifted one nucleotide
5′ to the preferred cleavage site when a noncleavable bond was placed at
the cleavage site. This was not observed with phosphorylated siRNA, sug-
gesting that the 5′ phosphate anchors the siRNA and target in the correct
position for cleavage.
Slicing cycle
Once RISC is loaded with the guide RNA, the complex is capable of mul-
tiple rounds of target binding and cleavage (Fig. 1a). RISC from D. mela-
nogaster cell extracts has an ATP-stimulated product-release step during
cycling71. Minimal recombinant RISC, composed of rHsAgo2 and an
siRNA, is capable of target cleavage but does not show ATP-stimulated
product release and seems to turn over very slowly24. Thus, ATP-depen-
dent product release does not reside in HsAgo2 and must be a feature of
another component of RISC. This observation is consistent with the lack
of ATP binding sites in the crystal structure of Argonaute.
Although an RNA duplex can be modeled into the crystal structure of
PfAgo, the fit is snug24. It is plausible that the substrate loading and turn-
over that are stimulated by ATP binding are dependent on a hinge move-
ment that opens the groove for substrate binding and release and closes
the groove for catalysis. This is consistent with normal mode analysis of
PfAgo, in which one of the two major motions is that of the PAZ domain
relative to the remaining portions of PfAgo, which results in opening and
widening of the RNA binding groove72. Movement of the PAZ domain
is also consistent with the slight rotation of the PAZ domain observed
in the crystal structure of AaAgo relative to the PAZ domain in PfAgo17,
which suggests that the orientation of the PAZ domain is relatively flex-
ible within Argonaute. The second major motion observed in the normal
mode analysis is a hinge motion around the smaller positively charged
groove between the N-terminal and PIWI domains.
A separate purification of human RISC containing Dicer, TRBP
and Argonaute 2 was capable of pre-microRNA cleavage, guide-strand
loading and multiple rounds of mRNA target cleavage73. Interestingly,
this study clearly showed that turnover is stimulated by nucleotides
and nucleotide analogs but (importantly) does not require nucleotide
hydrolysis. Furthermore, it seems that the minimal RNAi machinery
is composed of these three proteins, although low concentrations of
copurifying factors cannot be excluded.
H3K9me2
Pol II
Rik1
5' RdRC
RITS
Ago 5'
Dicer
Figure 4 Model for the role of slicing in TGS in S. pombe. RNA polymerase II
(Pol II) nascent transcripts are targeted by the small RNA–containing RITS
complex. Slicing by Ago results in a free 3′-OH in the transcript, creating a
substrate for the RdRC. RdRC converts the nascent transcript into double-
stranded RNA, which is a substrate for Dicer. Dicer processing produces
secondary siRNAs complementary to the region to be silenced that are
incorporated into RITS for further targeting. Then, the Rik1 complex, which
includes the histone methyltransferase Clr4, results in histone H3 Lys9
dimethylation (H3K9me2), which leads to the spreading of silencing.
41
 REVIEW
Slicing and TGS
Although the role of slicing in post-transcriptional gene silencing has
been established, it has recently been shown that slicing is also required
for transcriptional gene silencing43 (Fig. 4). Heterochromatic repeats
in S. pombe are transcriptionally silenced via histone H3 Lys9 dimeth-
ylation and the fission yeast heterochromatin protein 1 counterpart,
Swi674,75. Silencing is dependent on several multiprotein complexes:
the RITS complex, the Rik1-containing complex and the RNA-depen-
dent RNA polymerase (RdRP) complex. In addition to SpAgo1, the
RITS complex contains two other proteins—a chromodomain protein
(Chp1) and a protein of unknown function (Tas3)8. The Rik1 complex
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
contains several proteins, including the histone methyltransferase Clr4,
which methylates histone H376–79. At heterochromatic repeats, genes
located downstream of the promoter of the silenced loci are silenced
more efficiently than those located upstream, which suggests a role
for transcription in spreading of silencing43. The siRNA-directed RITS
complex recruits RdRP to nascent transcripts, where RdRP converts the
transcribed RNA into double-stranded RNA80. This double-stranded
RNA is then the substrate for Dicer, which produces siRNAs that are
complementary to the transcribed loci. Incorporation of newly formed
siRNAs into RITS is thought to result in degradation of transcribed
RNA via slicing; it is also thought to direct the Rik1 complex in facili-
tating the spread of histone methylation, which in turn leads to the
spreading of silencing.
Mutation of the active site residues of SpAgo1 results in a derepres-
sion of silencing at repeats, accompanied by a reduction in histone H3
Lys9 dimethylation and an increase in histone H3 Lys4 methylation43.
Lower concentrations of RdRP, but not SpAgo1, are observed at repeats
in Slicer mutant strains, which suggests that slicing (and not localization
of SpAgo1) is required to recruit RdRP to these repeats. Furthermore,
transcripts from both strands of the silenced loci accumulate in mutant
strains, which indicates that these transcripts are the targets for the Slicer
SpAgo1. These results suggest that SpAgo1 slicing results in the produc-
tion of a free 3′-OH that is the target for RdRP activity, thereby initiating
the spreading of silencing as described above.
Concluding remarks
Though we have unraveled one of the key functions of Argonaute in
RNAi, we still do not fully understand what makes Argonaute an active
Slicer, apart from the required catalytic residues. What determines
whether a particular small RNA will mediate target degradation or
translational repression? Is the diversification of the Argonautes a con-
sequence of which small RNA they recruit? Is the conformation of the
protein dependent on the substrate it targets, or does the protein con-
formation dictate its targets? Further biochemical and structural studies
will no doubt shed some light on these outstanding questions.
ACKNOWLEDGMENTS
RNAi is a flourishing field with a great deal of wonderful studies, some of which
we were not able to cite in this short survey, and for that we apologize. This work
was supported by a grant from the US National Institutes of Health to L.J. (R01-
GM072659).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturechemicalbiology
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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