RESEARCH ARTICLES
`
`Crystal Structure of Argonaute
`and Its Implications for RISC
`Slicer Activity
`Ji-Joon Song,1,2 Stephanie K. Smith,2 Gregory J. Hannon,1
`Leemor Joshua-Tor1,2*
`
`Argonaute proteins and small interfering RNAs (siRNAs) are the known signature
`components of the RNA interference effector complex RNA-induced silencing
`complex (RISC). However, the identity of “Slicer,” the enzyme that cleaves the
`messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we
`report the crystal structure of the Argonaute protein from Pyrococcus furiosus at
`2.25 angstrom resolution. The structure reveals a crescent-shaped base made up
`of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ)
`domain is held above the base by a “stalk”-like region. The PIWI domain (named for
`the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-
`aspartate-glutamate motif, strongly implicating Argonaute as “Slicer.” The architecture
`of the molecule and the placement of the PAZ and PIWI domains define a groove for
`substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.
`
` on July 2, 2009
`
`www.sciencemag.org
`
`Downloaded from
`
`Overall architecture. The structure of
`the full-length Argonaute from the archaebac-
`terium P. furiosus (PfAgo) was determined by
`x-ray crystallography to 2.25 Å resolution (ta-
`ble S1). The N-terminal, middle, and PIWI do-
`mains form a crescent-shaped base, with the PIWI
`domain at the center of the crescent. The region
`following the N-terminal domain forms a “stalk”
`that holds the PAZ domain above the crescent and
`an interdomain connector cradles the four do-
`mains of the molecule (Fig. 1). This architecture
`forms a groove at the center of the crescent and
`the PAZ domain closes off the top of this groove.
`The N-terminal domain consists of a long
`strand at the bottom of the crescent, followed by
`a region of a small four-stranded ␤ sheet, three
`␣ helices, and a ␤ hairpin, which then extends
`to the three-stranded antiparallel ␤ sheet stalk.
`The PAZ domain (residues 152 to 275) is a
`globular domain that adopts an OB-like ␤
`barrel fold with an attachment of two ␣ helices
`on one side of the barrel and a cleft in between.
`This cleft is angled toward the crescent. The
`middle domain (residues 362 to 544) is an ␣/␤
`open sheet domain composed of a central
`three-stranded parallel ␤ sheet surrounded by ␣
`helices. This domain is similar to the glucose-
`galactose-arabinose-ribose–binding protein fami-
`ly and is most similar to Lac repressor (15). The
`middle domain also has a small three-stranded ␤
`sheet on the outer surface of the crescent, connect-
`ing it to the rest of the molecule.
`The PIWI domain, which is at the C termi-
`nus of Argonaute (residues 545 to 770), is the
`most surprising portion of the structure, as we
`describe below. It sits in the middle of the
`
`with the 3⬘ ends of siRNAs in the two
`proteins containing this domain, Dicer and
`Argonaute (10). In RISC, the Argonaute PAZ
`domain would hold the 3⬘ end of the single-
`stranded siRNA, perhaps orienting recognition
`and cleavage of mRNA substrates. However, the
`nuclease responsible for cleavage, dubbed “Slic-
`er,” has so far escaped identification.
`In an effort to deepen our understanding
`of the role of Argonaute proteins in RNAi, we
`have conducted structural studies of a full-
`length Argonaute protein from P. furiosus.
`
`RNA interference (RNAi) is triggered by the
`presence of double-stranded RNA (dsRNA)
`(1). A ribonuclease (RNase) III family enzyme,
`Dicer, initiates silencing by releasing ⬃20 base
`duplexes, with two-nucleotide 3⬘ overhangs
`called siRNAs (2, 3). The RNAi pathway also
`mediates the function of endogenous, noncod-
`ing regulatory RNAs called microRNAs
`(miRNAs) [reviewed in (4)]. Both miRNAs
`and siRNAs guide substrate selection by similar
`if not identical effector complexes called RISC
`(4). These contain single-stranded versions of
`the small RNA and additional protein compo-
`nents (5–7). Of those, the signature element,
`which virtually defines a RISC, is a member of
`the Argonaute family of proteins (8).
`Argonaute proteins are defined by the
`presence of PAZ and PIWI domains (9).
`Recent structural and biochemical analyses of
`the PAZ domain have begun to reveal Argo-
`naute as the protein that interacts directly
`with the small RNA in RISC (10–14). The
`PAZ domain forms a deviant oligonucleotide/
`oligosaccharide-binding (OB) fold containing
`a central cleft lined with conserved aromatic
`residues that bind specifically to single-
`stranded 3⬘ ends (10, 12). This was confirmed
`by subsequent structural studies of PAZ com-
`plexed with nucleic acids (13, 14). On the
`basis of these studies, we first proposed a
`model in which the PAZ domain interacts
`
`1Watson School of Biological Sciences, 2Keck Struc-
`tural Biology Laboratory, Cold Spring Harbor Labora-
`tory, 1 Bungtown Road, Cold Spring Harbor, NY
`11724, USA.
`
`*To whom correspondence should be addressed. E-
`mail: leemor@cshl.edu
`
`Fig. 1. Crystal structure of P. furiosus Argonaute. (A) Stereoview ribbon representation of
`Argonaute showing the N-terminal domain (blue), the “stalk” (light blue), the PAZ domain (red),
`the middle domain (green), the PIWI domain (purple), and the interdomain connector (yellow).
`Active site residues are drawn in stick representation. Disordered loops are drawn as dotted lines.
`(B) Schematic diagram of the domain borders.
`
`1434
`
`3 SEPTEMBER 2004 VOL 305 SCIENCE www.sciencemag.org
`
`Alnylam Exh. 1047
`
`

`

` on July 2, 2009
`
`www.sciencemag.org
`
`Downloaded from
`
`R E S E A R C H A R T I C L E S
`
`Fig. 2. The PAZ domains of PfAgo and
`hAgo1 have very similar structures. (A)
`Stereoview diagram of the superposition
`of C␣ atoms from the PAZ domain of
`PfAgo (red) and the PAZ domain of hAgo1
`(gray). Dotted lines represent disordered
`regions. (B) Side chains involved in bind-
`ing the two-nucleotide 3⬘ overhang are
`shown in stick representation with PfAgo
`residues in atom colors (carbon, yellow;
`oxygen,
`red; and nitrogen, blue), and
`hAgo1 residues in green.
`
`Fig. 3. PIWI is an RNase H domain. (A) Ribbon diagrams of the PIWI domain, Escherichia coli RNase
`HI and Methanococcus jannaschii RNase HII. The three structures are shown in a similar view with
`the secondary structure elements of the canonical RNase H fold in color. Active site residues are
`shown in stick representation. (B) This view of the active site residues is rotated ⬃180° about the
`y axis compared with the view in (A). The Mg2⫹ ion in RNase HI is shown as a pink sphere. A strong
`difference electron density (⬎4.5␴) found in the active site of PIWI that was assigned as a water
`molecule is shown as a green sphere. Secondary structural elements of the RNase H fold are colored
`from red to pink (red, orange, yellow, green, blue, purple, pink) as ordered in the protein sequence.
`
`crescent and below the PAZ domain. The crys-
`tal structure reveals the presence of a prominent
`central five-stranded ␤ sheet flanked on both
`sides by ␣ helices. A smaller ␤ sheet extends
`from the central ␤ sheet and attaches PIWI to
`the N-terminal domain and to portions of the
`interdomain connector.
`The PAZ domain. The PAZ domain su-
`perimposes well with other PAZ domains that
`have known structures (10–12, 14), although
`the attachment in the archael protein has two ␣
`helices rather than an ␣ helix and a ␤ hairpin
`(Fig. 2A). Other differences lie in loop regions.
`The root mean square deviation (RMSD) be-
`tween human Argonaute-1 (hAgo1)–PAZ (14)
`and the PAZ domain in this structure is about
`1.4 Å (for 53 C␣’s). Despite close structural
`similarities, primary sequence comparisons
`failed to reveal a PAZ domain in PfAgo (fig.
`S1), whereas the presence and location of the
`PIWI domain was easily detected in Basic Lo-
`cal Alignment Search Tool (BLAST) searches.
`Importantly, conserved aromatic residues
`that bind the two-nucleotide 3⬘ overhang of
`an siRNA (10, 13, 14) are all present in
`PfAgo (Fig. 2B). Curiously, in some cases,
`these side chains occupy similar positions in
`space, although they are anchored to posi-
`tions on the peptide backbone differing from
`those in the eukaryotic proteins. Specifically,
`Y212, Y216, H217, and Y190 of PfAgo are
`equivalent to Y309, Y314, H269, and Y277,
`respectively, of hAgo1 (16), which bind the
`oxygens of the phosphate that links the two
`bases in the overhang. Residue Y190 of
`PfAgo superimposes perfectly on hAgo1-
`Y277, which binds the 2⬘ hydroxyl of the
`penultimate nucleotide. Residues L263 and
`I261 can assume the role of L337 and T335,
`which anchor the sugar ring of the terminal
`residue through van der Waals interactions.
`An aromatic residue, F292 in hAgo1 stacks
`against the terminal nucleotide. This position
`is occupied by another aromatic, W213, in
`PfAgo. Finally, R220 in our structure is posi-
`tioned similarly to K313 that contacts the
`penultimate nucleotide. Residues that bind
`other regions of the RNA include K191 (R278
`in hAgo1) and Y259 (K333 in hAgo1) to bind
`phosphates. Additional PAZ residues, such as
`K252, K248, Q276, and N176 are probably also
`used to bind that siRNA strand. We therefore
`reason that the PAZ domain in PfAgo binds
`RNA 3⬘ ends, as do PAZ domains of fly and
`human Argonautes.
`PIWI is an RNase H domain. The PIWI
`domain core has a tertiary structure belonging
`to the RNase H family of enzymes. This fold
`is also characteristic of other enzymes with
`nuclease or polynucleotidyl transferase activ-
`ities, such as human immunodeficiency virus
`and avian sarcoma virus integrases (17, 18),
`RuvC (a Holliday junction endonuclease)
`(19), and transposases such as Mu (20) and
`Tn5 (21). The closest matches, however, are
`
`www.sciencemag.org SCIENCE VOL 305 3 SEPTEMBER 2004
`
`1435
`
`

`

` on July 2, 2009
`
`www.sciencemag.org
`
`Downloaded from
`
`activity is another hallmark of RNase H en-
`zymes, a requirement that RISC shares (27).
`The PAZ domain, recognizing the 3⬘ ends of
`siRNAs, and the PIWI domain, now shown to
`be an RNase H domain, combine the necessary
`features of the slicing component of the RNAi
`machinery. Therefore, Argonaute, the signature
`component of RISC, appears to be Slicer itself.
`A model
`for
`siRNA-guided mRNA
`cleavage. The overall structure of Argonaute
`defines a distinct groove through the protein,
`which has a claw shape and bends between the
`PAZ and N-terminal domains. A notable fea-
`ture of the structure is evident when the elec-
`trostatic potential is mapped on the surface of
`the protein. As shown in Fig. 4A, the surface of
`this inner groove is lined with positive charges
`suitable for interaction with the negatively
`charged phosphate backbone and with the 2⬘
`hydroxyl moieties of RNA,
`implicating the
`groove for substrate binding.
`To examine possible substrate binding
`modes for Argonaute, we superimposed the
`PAZ domains from PfAgo and hAgo1 (14) and
`examined the position of the RNA in the hAgo1
`complex with respect to PfAgo. The strand that
`interacts with the PAZ cleft is the siRNA guide
`(Fig. 4B). Leaving the cleft, the RNA tracks the
`top of the PAZ ␤ barrel allowing similar, if not
`identical, interactions with the PfAgo PAZ, as
`observed in the hAgo1 PAZ-RNA complex. A
`long loop present in the PfAgo PAZ domain
`would probably move up slightly to accommo-
`date the siRNA. The other strand would repre-
`sent the mRNA substrate and would enter the
`binding groove with its 5⬘ end between the PAZ
`and N-terminal domains, with the latter acting
`as an “mRNA grip.” Another extension of the
`groove lies between the N-terminal and the
`PIWI domains and could accommodate a single-
`stranded nucleic acid. However, the function of
`this feature is presently difficult to predict.
`Extending the dsRNA further into the mol-
`ecule along the binding groove by model build-
`ing positions the mRNA above the active site
`located in the PIWI domain, nine nucleotides
`from the 5⬘ side end of the double-stranded
`region. On the basis of this model, the scissile
`bond falls between nucleotides 11 and 12,
`counting from the 3⬘ end of the guide. This
`precisely coincides with the demonstrated
`cleavage of mRNAs by RISC, 10 nucleotides
`from the 5⬘ end of a 21-nucleotide siRNA. The
`remainder of the RNA would continue along
`the binding groove (Fig. 4C). The length of the
`groove appears sufficient to accommodate the
`entire siRNA guide, with the 5⬘ end of the guide
`probably interacting with the other side of the
`groove to sense the siRNA 5⬘ phosphate. Ad-
`ditionally, from studies of other RNase H en-
`zymes, we expect Argonaute to sense the minor
`groove width of the dsRNA, which differs from
`that of dsDNA and from an RNA/DNA hybrid.
`Such a hypothesis is in accord with the inability
`of RISC to cut DNA substrates (26). Although
`
`R E S E A R C H A R T I C L E S
`
`with RNase HII (22) and RNase H1 (23). The
`domains are topologically identical: The
`RMSDs between RNase HII and PfAgo (for
`74 C␣’s) and between RNase HI and PfAgo
`(for 66 C␣’s) are both 1.9 Å (Fig. 3A). RNase
`H fold proteins all have a five-stranded mixed
`␤ sheet surrounded by helices. PIWI has an
`insertion between the last strand and the last
`helix of the RNase H fold that links it to the
`rest of the protein.
`All of these enzymes contain three highly
`conserved catalytic carboxylates, which com-
`prise the DDE motif (24). Two of these side
`chains are always located on the first strand, ␤1,
`which is the central strand of the ␤ sheet, and at
`the C terminus of the fourth strand, ␤4, of the
`RNase H fold, which is adjacent to ␤1. The
`position of the third carboxylate varies. Nota-
`bly, two aspartate residues in PIWI were locat-
`ed at the same positions as the invariant car-
`boxylates of the RNase H fold (Fig. 3B). These
`are D558, located on the first ␤ strand, and D628,
`located at the end of the fourth strand of the
`PIWI domain. The only requirement for the
`third variable carboxylate is a reasonable spatial
`position at the active site. E635 is in close prox-
`
`imity to the two aspartates and we suggest that
`this glutamate serves as the third active-site
`residue. This residue is positioned on the sec-
`ond helix of the RNase H fold of PIWI (the blue
`helix in Fig. 3). These three residues are almost
`completely invariant in the 136 Argonaute pro-
`tein sequences examined (fig. S2). Interesting-
`ly, an arginine, R627, is also positioned at the
`center of the active site, as in the case of the IS4
`family of transposases such as Tn5, which ap-
`pear to have a DDRE motif (21). The active site
`is thus positioned in a cleft in the middle of the
`crescent in the groove below the PAZ domain.
`Ago as Slicer. The observation that the
`PIWI domain in Argonaute is an RNase H
`domain immediately implicated Argonaute as
`Slicer, the enzyme in RISC that cleaves the
`mRNA. RNase H enzymes cleave single-
`stranded RNA “guided” by the DNA strand in
`an RNA/DNA hybrid. Similarly, Argonautes
`might specialize in RNA cleavage, guided by
`the siRNA strand in a dsRNA substrate. More-
`over, RNase H enzymes produce products with
`3⬘-OH and 5⬘ phosphate groups (25), in agree-
`ment with the products of mRNA cleavage by
`RISC (26, 27). A dependence on Mg2⫹ for
`
`Fig. 4. A model for siRNA-guided
`mRNA cleavage by Argonaute.
`(A) View of the electrostatic sur-
`face potential of PfAgo indicat-
`ing a positively charged groove
`(blue). The approximate location
`of the active site is marked by a
`yellow asterisk. This view is
`slightly tilted on the horizontal
`axis compared to the view in Fig.
`1. Two of the loops were re-
`moved for a better view of the
`groove. (B) A 3⬘ portion of the
`siRNA (purple) was placed by su-
`perposition of the PAZ domain
`of the hAgo1-PAZ domain RNA
`complex on the PAZ domain of
`PfAgo. The passenger strand of the hAgo1-PAZ complex placed in a similar manner was used to
`model the mRNA strand (light blue) by extending the RNA two nucleotides at the 5⬘ end, and from
`the middle of that strand along the binding groove toward the active site in PIWI. The phosphate
`between nucleotides 11 and 12 from the 5⬘ end of the mRNA falls near the active site residues
`(red). The view is similar to the view in Fig. 1. (C) Schematic depiction of the model for
`siRNA-guided mRNA cleavage. The domains are colored as in Fig. 1. The siRNA (yellow) binds with
`its 3⬘ end in the PAZ cleft and the 5⬘ is predicted to bind near the other end of the cleft. The mRNA
`(brown) comes in between the N-terminal and PAZ domains and out between the PAZ and middle
`domain. The active site in the PIWI domain (shown as scissors) cleaves the mRNA opposite the
`middle of the siRNA guide.
`
`1436
`
`3 SEPTEMBER 2004 VOL 305 SCIENCE www.sciencemag.org
`
`

`

` on July 2, 2009
`
`www.sciencemag.org
`
`Downloaded from
`
`7. J. Martinez, A. Patkaniowska, H. Urlaub, R. Luhrmann,
`T. Tuschl, Cell 110, 563 (2002).
`8. S. M. Hammond, E. Bernstein, D. Beach, G. J. Hannon,
`Nature 404, 293 (2000).
`9. L. Cerutti, N. Mian, A. Bateman, Trends Biochem. Sci.
`25, 481 (2000).
`10. J. J. Song et al., Nature Struct. Biol. 10, 1026 (2003).
`11. K. S. Yan et al., Nature 426, 468 (2003).
`12. A. Lingel, B. Simon, E. Izaurralde, M. Sattler, Nature
`426, 465 (2003).
`13. A. Lingel, B. Simon, E. Izaurralde, M. Sattler, Nature
`Struct. Mol. Biol. 11, 576 (2004).
`14. J. B. Ma, K. Ye, D. J. Patel, Nature 429, 318 (2004).
`15. A. M. Friedman, T. O. Fischmann, T. A. Steitz, Science
`268, 1721 (1995).
`16. Single-letter abbreviations for the amino acid residues are
`as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,
`His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;
`S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
`17. F. Dyda et al., Science 266, 1981 (1994).
`18. J. Lubkowski et al., Biochemistry 38, 13512 (1999).
`19. M. Ariyoshi et al., Cell 78, 1063 (1994).
`20. P. Rice, K. Mizuuchi, Cell 82, 209 (1995).
`21. D. R. Davies, I. Y. Goryshin, W. S. Reznikoff, I. Ray-
`ment, Science 289, 77 (2000).
`22. L. Lai, H. Yokota, L. W. Hung, R. Kim, S. H. Kim, Struct.
`Fold Des. 8, 897 (2000).
`23. K. Katayanagi, M. Okumura, K. Morikawa, Proteins 17,
`337 (1993).
`24. W. Yang, T. A. Steitz, Structure 3, 131 (1995).
`25. U. Wintersberger, Pharmacol. Ther. 48, 259 (1990).
`
`R E S E A R C H A R T I C L E S
`
`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).
`29. J. W. Pham, J. L. Pellino, Y. S. Lee, R. W. Carthew, E. J.
`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
`
`www.sciencemag.org SCIENCE VOL 305 3 SEPTEMBER 2004
`
`1437
`
`