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`R E S E A R C H A R T I C L E S
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`26. J. Martinez, T. Tuschl, Genes Dev. 18, 975 (2004).
`27. D. S. Schwarz, Y. Tomari, P. D. Zamore, Curr. Biol. 14,
`787 (2004).
`28. Y. Tomari et al., Cell 116, 831 (2004).
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`Sontheimer, Cell 117, 83 (2004).
`30. J. Liu et al., Science 305, 1437 (2004); published
`online 29 July 2004 (10.1126/science.1102513).
`31. We thank E. Enemark for help with data collection, R.
`Martienssen and members of the Joshua-Tor labora-
`tory for helpful discussions, and M. Becker (beamline
`X25) for support with data collection at the National
`Synchrotron Light Source (NSLS). The NSLS is sup-
`ported by the U.S. Department of Energy, Division of
`Material Sciences and Division of Chemical Sciences.
`J.-J.S. is a Bristol-Myers Squibb Predoctoral Fellow.
`Coordinates have been deposited in the Protein Data
`Bank (accession code 1U04).
`
`Supporting Online Material
`www.sciencemag.org/cgi/content/full/1102514/DC1
`Materials and Methods
`Figs. S1 to S3
`Table S1
`References
`
`8 July 2004; accepted 21 July 2004
`Published online 29 July 2004;
`10.1126/science.1102514
`Include this information when citing this paper.
`
`Argonaute2 Is the Catalytic
`Engine of Mammalian RNAi
`Jidong Liu,1* Michelle A. Carmell,1,2* Fabiola V. Rivas,1
`Carolyn G. Marsden,1 J. Michael Thomson,3 Ji-Joon Song,1
`Scott M. Hammond,3 Leemor Joshua-Tor,1 Gregory J. Hannon1†
`
`Gene silencing through RNA interference (RNAi) is carried out by RISC, the
`RNA-induced silencing complex. RISC contains two signature components,
`small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we
`show that the multiple Argonaute proteins present in mammals are both
`biologically and biochemically distinct, with a single mammalian family
`member, Argonaute2, being responsible for messenger RNA cleavage ac-
`tivity. This protein is essential for mouse development, and cells lacking
`Argonaute2 are unable to mount an experimental response to siRNAs.
`Mutations within a cryptic ribonuclease H domain within Argonaute2, as
`identified by comparison with the structure of an archeal Argonaute protein,
`inactivate RISC. Thus, our evidence supports a model in which Argonaute
`contributes “Slicer” activity to RISC, providing the catalytic engine for RNAi.
`
`The presence of double-stranded RNA
`(dsRNA) in most eukaryotic cells provokes a
`sequence-specific silencing response known
`as RNA interference (RNAi) (1, 2). The
`dsRNA trigger of this process can be derived
`from exogenous sources or transcribed from
`endogenous noncoding RNA genes that pro-
`duce microRNAs (miRNAs) (1, 3).
`
`RNAi begins with the conversion of
`dsRNA silencing triggers into small RNAs
`of ⬃21 to 26 nucleotides (nts) in length (4).
`This is accomplished by the processing of
`triggers by specialized ribonuclease III
`(RNase III)–family nucleases, Dicer and
`Drosha (5, 6). Resulting small RNAs join
`an effector complex, known as RISC
`(RNA-induced silencing complex) (7 ). Si-
`lencing by RISC can occur through several
`mechanisms. In flies, plants, and fungi,
`dsRNAs can trigger chromatin remodeling
`and transcriptional gene silencing (8–11).
`RISC can also interfere with protein syn-
`thesis, and this is the predominant mecha-
`nism used by miRNAs in mammals (12,
`13). However,
`the best studied mode of
`
`a single-stranded RNA should bind fairly readi-
`ly, opening the claw of the molecule might
`assist binding the mRNA, after which Argo-
`naute might close on the double-stranded sub-
`strate. A possible hinge region exists in the
`interdomain connector at residues 317 to 320.
`This hinge could lift the PAZ away from the
`crescent base, perhaps allowing the RISC load-
`ing complex to assist in assembling an active
`complex (28, 29).
`The notion that RISC “Slicer” activity resides
`in Argonaute itself was tested in a mammalian
`system, by mutational analysis of hAgo2 (30).
`Conserved active site aspartates in hAgo2 were
`altered and the mutants lost nuclease activity but
`retained siRNA binding. This supports the model
`in which Argonaute itself functions as the Slicer
`enzyme in the RNAi pathway.
`Many questions regarding the details of
`the mechanism for siRNA-guided mRNA
`cleavage remain. Several Argonaute protein
`family members appear to be inactive toward
`mRNA cleavage despite the presence of the
`catalytic residues. This situation might be
`analogous to the case of the Tn5 transposase
`and its inhibitor, which possess a catalytic
`domain with an RNase H–like fold. Tn5 in-
`hibitor is a truncated version of the active
`Tn5 transposase and retains essential catalyt-
`ic residues. However, there are major confor-
`mational differences between the two (21).
`Mutations have been introduced into a cata-
`lytically active Ago protein, hAgo2, in the
`vicinity of the active site, which change res-
`idues to corresponding residues in an inactive
`Ago, hAgo1. These inactivate Ago2 for
`cleavage, indicating that there are determi-
`nants for catalysis beyond simply the catalyt-
`ic triad and that relatively minor alterations in
`the PIWI domain can have profound effects
`on its activity toward RNA substrates. In
`addition, interactions with other factors may
`be needed to create a fully active Slicer. The
`common fold in the catalytic domain of Ar-
`gonaute family members and transposases
`and integrases is also intriguing given the
`relationship of RNAi with control of transpo-
`sition. Notably, the identification of the cat-
`alytic center of RISC awaited a drive toward
`understanding RNAi at a structural
`level.
`Thus, it seems likely that, as in the present
`example, a full understanding of the underly-
`ing mechanism of RNAi will derive from a
`combination of detailed biochemical and
`structural studies of RISC.
`
`References and Notes
`1. A. Fire et al., Nature 391, 806 (1998).
`2. S. M. Elbashir, J. Martinez, A. Patkaniowska, W. Len-
`deckel, T. Tuschl, EMBO J. 20, 6877 (2001).
`3. E. Bernstein, A. A. Caudy, S. M. Hammond, G. J.
`Hannon, Nature 409, 363 (2001).
`4. D. P. Bartel, Cell 116, 281 (2004).
`5. A. Nykanen, B. Haley, P. D. Zamore, Cell 107, 309 (2001).
`6. D. S. Schwarz, G. Hutvagner, B. Haley, P. D. Zamore,
`Mol. Cell 10, 537 (2002).
`
`1Cold Spring Harbor Laboratory, Watson School of
`Biological Sciences, 1 Bungtown Road, Cold Spring
`Harbor, NY 11724, USA. 2Program in Genetics, Stony
`Brook University, Stony Brook, NY 11794, USA. 3De-
`partment of Cell and Developmental Biology, Univer-
`sity of North Carolina, Chapel Hill, NC 27599, USA.
`
`*These authors contributed equally to this work.
`†To whom correspondence should be addressed. E-
`mail: hannon@cshl.edu
`
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`R E S E A R C H A R T I C L E S
`
`RISC action is mRNA cleavage (14, 15 ).
`When programmed with a small RNA that
`is fully complementary to the substrate
`RNA, RISC cleaves that RNA at a discrete
`position, an activity that has been attributed
`to an unknown RISC component, “Slicer”
`(16, 17 ). Whether or not RISC cleaves a
`substrate can be determined by the degree
`of complementarity between the siRNA and
`mRNA, as mismatched duplexes are often
`not processed (16). However, even for
`mammalian miRNAs, which normally re-
`press at
`the level of protein synthesis,
`cleavage activity can be detected with a
`substrate that perfectly matches the miRNA
`sequence (18). This result prompted the
`hypothesis that all RISCs are equal, with
`the outcome of the RISC-substrate interac-
`tion being determined largely by the char-
`acter of the interaction between the small
`RNA and its substrate.
`RISC contains two signature components.
`The first is the small RNA, which cofraction-
`ated with RISC activity in Drosophila S2 cell
`extracts (7), and whose presence correlated
`with dsRNA-programmed mRNA cleavage
`in Drosophila embryo lysates (14, 15). The
`second is an Argonaute (Ago) protein, which
`was identified as a component of purified
`RISC in Drosophila (19). Subsequent studies
`have suggested that Argonautes are also key
`components of RISC in mammals, fungi,
`worms, protozoans, and plants (17, 20).
`Argonautes are often present as multi-
`protein families and are identified by two
`characteristic domains, PAZ and PIWI
`(21). These proteins mainly segregate into
`two subfamilies, comprising those that are
`more similar to either Arabidopsis Argo-
`naute1 or Drosophila Piwi. The Argonaute
`family was first linked to RNAi through
`genetic studies in Caenorhabditis elegans,
`which identified Rde-1 as a gene essential
`for silencing (22). Our subsequent place-
`ment of a Drosophila Argonaute protein in
`RISC (19) prompted us to explore the roles
`of this protein family. Toward this end, we
`have undertaken both biochemical and ge-
`netic studies of the Ago1 subfamily pro-
`teins in mammals.
`Mammals contain four Argonaute1 sub-
`family members, Ago1 to Ago4 [nomencla-
`ture as in (23); see fig. S1]. We have
`previously shown that different Argonaute
`family members in Drosophila preferential-
`ly associate with different small RNAs,
`with Ago1 preferring miRNAs and Ago2
`siRNAs (24 ). Recent studies of Drosophila
`melanogaster (dm) Ago1 and dmAgo2 mu-
`tants have strengthened these conclusions
`(25 ). To assess whether mammalian Ago
`proteins specialized in their interactions
`with small RNAs, we examined Ago-
`associated miRNA populations by microar-
`ray analysis. Ago1-, Ago2- and Ago3-
`
`associated RNAs were hybridized to mi-
`croarrays that report the expression status
`of 152 human microRNAs. Patterns of
`associated RNAs were identical within ex-
`perimental error in each case (Fig. 1A).
`Additionally, each of the tagged Ago pro-
`teins associated similarly with a cotrans-
`fected siRNA (Fig. 1C).
`Previous studies have used tagged siRNAs
`to affinity purify Argonaute-containing
`RISC (17 ). These preparations, containing
`mixtures of at least two mammalian Argo-
`nautes, were capable of cleaving synthetic
`mRNAs that were complementary to the
`tagged siRNA. We examined the ability of
`purified complexes containing individual
`Argonaute proteins to catalyze similar
`cleavages. Unexpectedly,
`irrespective of
`the siRNA sequence, only Ago2-containing
`RISC was able to catalyze cleavage (Fig.
`1B and fig. S2). All three Ago proteins were
`similarly expressed and bound similar amounts of
`transfected siRNA (Fig. 1, C and D).
`These results demonstrated that mam-
`malian Argonaute complexes are biochem-
`ically distinct, with only a single family
`member being competent for mRNA cleav-
`age. To examine the possibility that Ago
`proteins might also be biologically special-
`ized, we disrupted the mouse Ago2 gene by
`targeted insertional mutagenesis (fig. S3
`and Fig. 2A) (26 ). Intercrosses of Ago2
`heterozygotes produced only wild-type and
`
`heterozygous offspring, strongly suggest-
`ing that disruption of Ago2 produced an
`embryonic-lethal phenotype.
`Ago2-deficient mice display several de-
`velopmental abnormalities beginning ap-
`proximately halfway through gestation.
`Both gene-trap and in situ hybridization
`data of day 9.5 embryos show broad ex-
`pression of Ago2 in the embryo, with some
`hot spots of expression in the forebrain,
`heart, limb buds, and branchial arches (Fig.
`2, F and G). The most prominent phenotype
`is a defect in neural tube closure (Fig. 2, D
`and E), often accompanied by apparent
`mispatterning of anterior structures, includ-
`ing the forebrain (Fig. 2, C and D). Rough-
`ly half of the embryos display complete
`failure of neural tube closure in the head
`region (Fig. 2E), while all embryos display
`a wavy neural tube in more caudal regions.
`Mutant embryos also suffer from apparent
`cardiac failure. The hearts are enlarged and
`often accompanied by pronounced swelling
`of the pericardial cavity (Fig. 2C). By day
`10.5, mutant embryos are severely devel-
`opmentally delayed compared with wild-
`type and heterozygous littermates (Fig.
`2B). This large difference in size, like the
`apparent cardiac failure, may be accounted
`for by a general nutritional deficiency
`caused by yolk sac and placental defects
`(27 ), as histological analysis reveals abnor-
`malities in these tissues.
`
`Fig. 1. Only mammalian Ago2
`can form cleavage-competent
`RISC. (A) The miRNA popula-
`tions
`associated with Ago1,
`Ago2, and Ago3 were measured
`by microarray analysis as de-
`scribed in (44). The heat map
`shows normalized log-ratio val-
`ues for each data set, with yel-
`low representing increased rela-
`tive amounts and blue indicating
`decreased amounts relative to
`the median. The top 25 log ra-
`tios are shown in the expanded
`region. In each panel, “control”
`indicates parallel analysis of cells
`transfected with a vector con-
`trol. (B) The 293T cells were
`transfected with a control vector
`or with vectors encoding myc-
`tagged Ago1, Ago2, or Ago3,
`along with an siRNA that targets
`firefly luciferase.
`Immunopre-
`cipitates were tested for siRNA-
`directed mRNA cleavage as de-
`scribed in (44). Positions of 5⬘
`and 3⬘ cleavage products are
`shown. (C) Immunoprecipitates
`as in (B) were tested for in vivo
`siRNA binding by Northern blot-
`ting of Ago immunoprecipitates
`(44). (D) Western blots of trans-
`fected cell lysates show similar
`levels of expression for each re-
`combinant Argonaute protein.
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`R E S E A R C H A R T I C L E S
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`Evolutionary conservation of an essential
`cleavage-competent RISC in organisms in
`which miRNAs predominantly act by trans-
`lational regulation raises the possibility that
`target cleavage by mammalian miRNAs
`might be more important and widespread
`than previously appreciated.
`Numerous studies have indicated that
`experimentally triggered RNAi in mamma-
`lian cells proceeds through siRNA-directed
`mRNA cleavage because in many, but not
`all, cases, reiterated binding sites are nec-
`essary for repression at the level of protein
`synthesis [see, for example (13, 32, 33)]. If
`Ago2 were uniquely capable of assembling
`into
`cleavage-competent
`complexes
`in
`mice, then embryos or cells lacking Ago2
`might be resistant to experimental RNAi.
`To address this question, we prepared
`mouse embryo fibroblasts (MEFs)
`from
`E10.5 embryos from Ago2 heterozygous
`intercrosses. Reverse transcription poly-
`merase chain reaction (RT-PCR) analysis
`and genotyping revealed that we were able
`to obtain wild-type, mutant, and heterozy-
`gous MEF populations. Importantly, MEFs
`also express other Ago proteins, including
`Ago1 and Ago3 (Fig. 3A). Ago2-null MEFs
`were unable to repress gene expression in
`response to an siRNA (Fig. 3B and fig. S5).
`This defect could be rescued by the addi-
`tion of a third plasmid that encoded human
`Ago2 but not by a plasmid encoding human
`Ago1 (Fig. 3B). In contrast, responses were
`intact for a reporter of repression at the
`level of protein synthesis, mediated by an
`siRNA binding to multiple mismatched
`sites (32) (Fig. 3C).
`Because Ago2 is exceptional in its abil-
`ity to form cleavage-competent complexes,
`we set out to map the determinants of this
`capacity. Deletion analysis indicated that
`an intact Ago2 was required for RISC ac-
`
`Not all Argonaute proteins are required
`successful mammalian development
`for
`(28, 29). Thus,
`it
`is unclear why Ago2
`should be required for development, while
`other Ago proteins are dispensable. Ago
`subfamily members are expressed in over-
`lapping patterns in humans (30). In situ
`hybridization demonstrates overlapping ex-
`pression patterns for Ago2 and Ago3 in
`mouse embryos (Fig. 2F and fig. S4). Con-
`
`sidered together with the essentially iden-
`tical patterns of miRNA binding, our re-
`sults suggest the possibility that the ability
`of Ago2 to assemble into catalytically ac-
`tive complexes might be critical for mouse
`development. Although most miRNAs reg-
`ulate gene expression at the level of protein
`synthesis, recently miR196 has been dem-
`onstrated to cleave the mRNA encoding
`HoxB8, a developmental regulator (31).
`
`Fig. 2. Argonaute2 is essential for
`mouse development.
`(A) Total
`RNA from wild-type or mutant
`embryos was tested for expression
`of Ago1, Ago2, or Ago3 by RT-PCR.
`Actin was also examined as a con-
`trol. (B) At day E10.5, Ago2-null
`embryos show severe develop-
`mental delay as compared with
`heterozygous and wild-type litter-
`mates. These embryos also show a
`variety of developmental defects,
`including swelling inside the peri-
`cardial membrane (indicated by
`arrow) (h, heart) (C) and failure to
`close the neural tube (D and E).
`Arrows in (D) indicate the edges of
`the neural tube that has failed to
`close. In caudal regions, where the
`neural tube does close, it has an
`abnormal appearance, being wavy as compared with wild-type embryos (E) (compare wild-type and
`Ago2 ⫺/⫺). Ago2 is expressed in most tissues of the developing embryo as measured by in situ
`hybridization (F) or by analysis of an Ago2 gene-trap animal (G). In (F), f is forebrain, b is branchial
`arches, h is heart, and lb is limb bud, all of which are relative hot spots for Ago2 mRNA. In (G), the left
`embryo shows similar patterns when staining for the gene-trap marker, ␤-galactosidase, proceeds for
`only a short period. Longer incubation (G, right) gives uniform staining throughout the embryo.
`
`Fig. 3. Argonaute2 is essential
`for RNAi in MEFs. (A) RT-PCR
`of mRNA prepared from wild-
`type or Ago2⫺/⫺ MEFs reveals
`consistent expression of Ago1
`and Ago3 but a specific lack of
`Ago2 expression in the null
`MEF. Actin mRNA serves as a control. (B) Wild-type and mutant MEFs were
`cotransfected with plasmids encoding Renilla and firefly luciferases, either
`with or without firefly siRNA. Ratios of firefly to Renilla activity, normalized
`to 1 for the no-siRNA control, were plotted. For each genotype, the ability
`of Ago1 and Ago2 to rescue suppression was tested by cotransfection with
`expression vectors encoding each protein as indicated. (C) NIH-3T3 cells,
`
`wild-type MEFs, or Ago2 mutant MEFs were tested as described in (B)
`(except that Renilla/firefly ratios are plotted) for their ability to suppress a
`reporter of repression at the level of protein synthesis. In this case, the
`Renilla luciferase mRNA contains multiple imperfect binding sites for a
`CXCR4 siRNA. Cells were transfected with a mixture of firefly and Renilla
`luciferase plasmids with or without the siRNA.
`
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`R E S E A R C H A R T I C L E S
`
`tivity (fig. S6). We therefore used the se-
`quence of highly conserved but cleavage-
`incompetent Ago proteins as a guide to the
`construction of Ago2 mutants. A series of
`point mutations included H634P, H634A,
`Q633R, Q633A, H682Y, L140W, F704Y,
`and T744Y. Whereas all of these mutations
`retain siRNA-binding activity and most re-
`tain cleavage activity, changes at Q633 and
`H634 have a profound effect on target
`cleavage (Fig. 4). Both the Q633R and
`H634P mutations, in which residues were
`
`changed to corresponding residues in Ago1
`and Ago3, abolished catalysis. Changing
`H634 to A also inactivated Ago2, whereas
`a similar change, Q633A, was permissive
`for cleavage. Thus, even relatively conser-
`vative changes can negate the ability of
`Ago2 to form cleavage-competent RISC.
`Several possibilities could explain a
`lack of cleavage activity for Ago2 mutants.
`Such mutations could interfere with the
`proper
`folding of Ago2. However,
`this
`seems unlikely because those same residues
`
`Fig. 4. Mapping the re-
`quirements for assem-
`bly of cleavage-compe-
`tent RISC. Ago1, Ago2,
`or mutants of Ago2
`were
`expressed
`as
`myc-tagged fusion pro-
`teins in 293T cells. In all
`cases, expression con-
`structs were cotrans-
`fected with a luciferase
`siRNA. Western blot-
`ting indicated similar
`expression
`for
`each
`mutant.
`Immunopre-
`cipitates containing in-
`dividual proteins were
`tested for cleavage ac-
`tivity against a lucif-
`erase mRNA (44). Posi-
`tions of 5⬘ and 3⬘ cleav-
`age products are indi-
`cated. SiRNA binding
`was examined for each
`mutant by Northern
`blotting of immunopre-
`cipitates or by staining of immunoprecipitates with Sybr Gold (Molecular Probes, Eugene, Oregon).
`Representatives for these assays are shown. In no case did we detect a defect in interaction of mutants
`with siRNAs.
`
`presumably permit proper folding in close-
`ly related Argonaute proteins, and mutant
`Ago2 proteins retained the ability to inter-
`act with siRNAs. Alternatively, cleavage-
`incompetent Ago2 mutants could lose the
`ability to interact with the putative Slicer.
`Finally, Ago2 itself might be Slicer, with
`our conservative substitutions altering the
`active center of the enzyme in a way that
`prevents cleavage.
`The last possibility predicted that we
`might reconstitute an active enzyme with
`relatively pure Ago2 protein. We immuno-
`affinity purified Ago2 from 293T cells and
`attempted to reconstitute RISC in vitro.
`Incubation with the double-stranded siRNA
`produced no appreciable activity, whereas
`Ago2 could be successfully programmed
`with single-stranded siRNAs to cleave a
`complementary substrate (Fig. 5A). Forma-
`tion of the active enzyme was unaffected by
`first washing the immunoprecipitates with
`up to 2.5 M NaCl or 1 M urea. A 21-nt
`single-stranded DNA was unable to direct
`cleavage (Fig. 5A). Programming could be
`accomplished with different siRNAs that
`direct activity against different substrates
`(fig. S7). RISC is formed though a con-
`certed assembly process
`in which the
`RISC-loading complex (RLC) acts in an
`adenosine triphosphate (ATP)– dependent
`manner to place one strand of the small
`RNA into RISC (34–36 ). In vitro reconsti-
`tution occurs in the absence of ATP, which
`suggests that Ago2 could be programmed
`with siRNAs without a need for the normal
`assembly process (Fig. 5A). However, in
`vitro reconstitution of RISC still requires
`
`Fig. 5. Argonaute2 is
`a candidate for Slic-
`er. (A) Ago2 protein
`was immunoaffinity
`purified from tran-
`siently transfected
`293T cells. The prep-
`aration contained
`two major proteins
`(protein gel), in addi-
`tion to heavy and
`light chains. These
`were identified by
`mass spectrometry
`as Ago2 and HSP90.
`Immunoprecipitates
`were mixed (44) in
`vitro with single-
`or double-stranded
`siRNAs or with a 21-
`nt DNA having the
`same sequence as the
`siRNA. Reconstituted
`RISC was tested for
`cleavage activity with
`a uniformly labeled synthetic mRNA. Positions of 5⬘ and 3⬘ cleavage products
`are noted. Where indicated, the siRNA was not 5⬘ phosphorylated and, in one
`case, ATP was not added to the reconstitution reaction. (B) Ago2 or Ago2
`mutants were assembled into RISC in vivo by cotransfection with siRNAs,
`followed by immunoaffinity purification or by in vitro reconstitution, mixing
`
`affinity-purified proteins with single-stranded siRNAs. These mutants were
`tested for activity against a complementary mRNA substrate. 5⬘ and 3⬘
`cleavage products are as in (A). (C and D) Both mutant proteins were
`expressed at levels similar to wild-type Ago2 and bound siRNAs as readily.
`Ago2 (H634P) and Ago2 (Q633R) behave similarly in this assay.
`
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`the essential characteristics of an siRNA.
`For example, single-stranded siRNAs that
`lack a 5⬘ phosphate group cannot reconsti-
`tute an active enzyme.
`Although consistent with the possibility
`that the catalytic activity of RISC is carried
`within Ago2, these results do not rule out
`the possibility that a putative Slicer copu-
`rifies with Ago2. To demonstrate more
`conclusively that Ago2 is Slicer, we turned
`to the crystal structure of an Argonaute
`protein from an archebacterium, Pyrococ-
`cus furiosus (37 ). This structure revealed
`that the PIWI domain folds into a structure
`analogous to the catalytic domain of RNase
`H and avian sarcoma virus (ASV) inte-
`grase. The notion that such a domain would
`lie at the center of RISC cleavage is con-
`sistent with previous observations. RNase
`H and integrases cleave their substrates,
`leaving 5⬘ phosphate and 3⬘ hydroxyl
`groups through a metal-catalyzed cleavage
`reaction (38, 39). Notably, previous studies
`have strongly indicated that
`the scissile
`phosphate in the targeted mRNA is cleaved
`via a metal ion in RISC to give the same
`phosphate polarity (40). Our in vitro data
`are consistent with the reconstituted RISC
`also requiring a divalent metal (fig. S8).
`The active center of RNase H and its
`relatives consists of a catalytic triad of
`three carboxylate groups contributed by as-
`partic or glutamic acid (38, 39). These ami-
`no acid residues coordinate the essential
`metal and activate water molecules for nu-
`cleolytic attack. Reference to the known
`structure of RNase H reveals two aspartate
`residues in the archeal Ago protein present
`at the precise spatial locations predicted for
`formation of an RNase H–like active site
`(37 ). These align with identical residues in
`the human Ago2 protein (fig. S9). There-
`fore, to test whether the PIWI domain of
`Ago2 provides catalytic activity to RISC,
`we changed the two conserved aspartates,
`D597 and D669, to alanine, with the pre-
`diction that either mutation would inacti-
`vate RISC cleavage. Consistent with our
`hypothesis, the mutant Ago2 proteins were
`incapable of assembling into a cleavage-
`competent RISC in vitro or in vivo, de-
`spite retaining the ability to bind siRNAs
`(Fig. 5, B to D).
`Considered together, our data provide
`strong support for the notion that Argonaute
`proteins are the catalytic components of
`RISC. First, the ability to form an active
`enzyme is restricted to a single mammalian
`family member, Ago2. This conclusion is
`supported both by biochemical analysis and
`by genetic studies in mutant MEFs. Second,
`single amino acid substitutions within Ago2
`that convert residues to those present
`in
`closely related proteins negate RISC cleav-
`age. Third, the structure of the P. furiosis
`
`Argonaute protein reveals provocative struc-
`tural similarities between the PIWI domain
`and the RNase H domains, providing a hy-
`pothesis for the method by which Argonaute
`cleaves its substrates. We tested this hypoth-
`esis by introducing mutations in the predicted
`Ago2 active site. It is extremely unlikely that
`such mutations could affect interactions with
`other proteins, because they are buried within
`a cleft of Ago.
`Our studies indicate that the Argonaute
`proteins that are unable to form cleavage-
`competent RISC differ from Ago2 at key
`positions that do not
`include the putative
`metal-coordinating
`residues
`themselves.
`However, we cannot yet, based either on
`biochemical or structural studies, provide a
`precise explanation for the catalytic defects in
`these proteins. It is conceivable that Ago1
`and Ago3 fail to coordinate the catalytic met-
`al or that the structure of the active site is
`distorted sufficiently that a bound metal is
`unable to access the scissile phosphate. Al-
`ternatively, catalytic mechanisms with two
`metal ions have been proposed for RNase H
`(38, 39), which leaves open the possibility
`that catalytically inert Ago family members
`might lack structures essential to bind the
`second metal ion.
`The relationship between the nuclease
`domain in PIWI and conserved nuclease
`domains in viral
`reverse transcriptases,
`transposases, and viral integrases has po-
`tential evolutionary implications. In Dro-
`sophila, plants, and C. elegans, the RNAi
`pathway has a major role in controlling
`parasitic nucleic acids such as viruses and
`transposons (41–43). The fact
`that
`the
`RNAi machinery shares a core structural
`domain with viruses and transposons sug-
`gests that this nucleic acid immune system
`may have arisen in part by pirating compo-
`nents from the replication and movement
`machineries of the very elements that RNAi
`protects against. This hypothesis is made
`even more poignant by considering the role
`of RNA-dependent RNA polymerases in
`RNAi, their functional relationship to viral
`replicases, and the possibility that
`the
`siRNAs themselves might first have served
`as primers that enable such replicases to
`duplicate primordial genomes.
`
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