Argonaute and RNA — getting into the groove

Argonaute and RNA — getting into the groove Ji-Joon Song and Leemor Joshua-Tor RNAi has made an enormous impact on biology in a very short period of time. It became an extraordinarily useful and simple tool for gene silencing, even as its fascinating mechanism was gradually unraveling. Understanding the mechanism of RNAi-related pathways, including both transcriptional and posttranscriptional gene silencing and even processes such as DNA elimination in Tetrahymena, have benefited from an incredible marriage of genetics, biochemistry, molecular biology, bioinformatics and finally structural biology. Structural biology played a key role in deciphering the role that a central player — the Argonaute protein — has in all RNAi processes. Addresses Watson School of Biological Sciences and W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA Corresponding author: Joshua-Tor, Leemor ([email protected])

Current Opinion in Structural Biology 2006, 16:5–11 This review comes from a themed issue on Protein–nucleic acid interactions Edited by Gregory D Van Duyne and Wei Yang Available online 24th January 2006 0959-440X/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.01.010

Introduction When a double-stranded (ds)RNA trigger is introduced into the cell, the dsRNA is processed by an RNase III family enzyme called Dicer. This processing produces dsRNA fragments known as small interfering RNAs (siRNAs) that are
21 nucleotides in length. As a direct result of RNase III type processing, these fragments have a characteristic two-nucleotide overhang at both 30 ends and a phosphate moiety at the 50 ends. From within the cell, primary microRNAs (pri-miRNAs) are produced from transcripts that fold back on themselves into hairpin-like structures that are processed in the nucleus by the microprocessor, which consists of another RNase III enzyme called Drosha and an auxillary factor called Pasha or DGCR8. The pre-miRNAs are then exported from the nucleus into the cytoplasm by Exportin 5, and after that get processed further by Dicer into miRNAs that are also
21 nucleotides in length. These siRNAs or miRNAs are then incorporated in an asymmetric fashion into an effector complex called the ‘RNA-induced silencing complex’, or the ‘RISC’. In Drosophila a series of complexes that are www.sciencedirect.com

involved in loading the siRNAs or miRNAs into RISC have been characterized. RISC complexes are typified, or defined, by two components: an Argonaute protein and the siRNA or miRNA. The siRNA or miRNA guides the RISC complex to its target through base complementarity. The best-characterized RNAi pathway, and the one that is predominantly used when one sets out to use RNAi as a gene knockdown technology, results in an endonucleolytic cut in the mRNA target, thus preventing gene expression from moving forward. This cleavage is made ten bases from the point at which the 50 end of the guide siRNA hybridizes to the target messenger RNA (mRNA). The identity of the endonuclease, which was nicknamed ‘Slicer’, remained obscure before structural and biochemical studies that will be described below. In another pathway, the RISC complex silences gene expression by inhibiting translation without appreciable destruction of the mRNA. For a recent review see [1]. Two key proteins always play a role in RNAi type processes. These are Dicer, which works in the initiation stage to produce the siRNAs and miRNAs, and Argonaute, a protein that is central to the effector stage when the RISC complex is guided to its target and carries out, or promotes, silencing. Argonaute, the signature component of RISC, has two characteristic domains — the PAZ domain (named after three proteins that contain this domain: Piwi, Argonaute and Zwille), which it shares with Dicer, and a unique domain known as the PIWI domain [2]. Both the PAZ and PIWI domains appear to be restricted to proteins involved in RNAi-type processes. Argonaute proteins are divided into two subfamilies, those most similar to Arabidopsis Argonaute 1 (AGO1) and those most similar to Drosophila Piwi, but both subfamilies retain both the PAZ and PIWI domains [3]. In this review, we will discuss the contribution of structural biology towards understanding the mechanism of this increasingly important pathway. Many of these studies are focused on Argonaute. Although insights into the mechanism of Dicer cleavage were made based on bacterial RNase III [4,5], these will not be addressed in this review.

PAZ domain as an siRNA binding module The first structures of modules of the RNAi machinery are those of the PAZ domain. These provided important insights into their function, which was completely unknown beforehand. The PAZ domain consists of
130 amino acids and is found only in two protein Current Opinion in Structural Biology 2006, 16:5–11

6 Protein–nucleic acid interactions

families — Argonaute and Dicer. Interestingly, both are involved in RNAi. Crystal and solution structures of PAZ domains from several Argonaute proteins were solved as free forms [6–8] and in complex with nucleic acids [9,10]. The PAZ domain is composed of two subdomains that have a cleft in between. One subdomain consists of a fivestranded open b-barrel with two helices on one end of the barrel. The other subdomain is made up of a b-hairpin followed by an a-helix. The b-barrel subdomain is highly reminiscent of an OB fold (named for oligonucleotide– oligosaccharide binding) although it has a somewhat different topology to the classical OB fold. OB-fold domains have variety of functions, with a major one being single-stranded nucleic acid binding [11]. Indeed, the PAZ domain binds nucleic acids. This was shown by several different methods [6–8]. Although there are some discrepancies depending on the method used, several common features were observed. First, the PAZ domain of Argonaute binds nucleic acids with low affinity, which suggests that other portions of the RISC complex, or other domains of Argonaute itself, provide much of the binding affinity. Second, the PAZ domain binds preferentially to RNA over DNA [7,8]. Third, binding is not sequence-dependent. Fourth, for Argonautes, the presence of a phosphate at the 50 end of an siRNA does not appear to contribute much to binding to the PAZ domain. Lastly, and most importantly, the PAZ domain recognizes the 30 ends of single-stranded (ss)RNAs [6,7]. Mapping conserved amino acids on the surface of the Ago2-PAZ domain revealed that the intersubdomain cleft contained almost all of the conserved residues, some of which are invariant among most PAZ domains. Interestingly, many of the invariant residues are aromatic. Mutational analysis of the invariant residues showed that they are involved in siRNA binding [6–8]. Considering that those residues are also conserved in Dicer PAZ domain, it is likely that the PAZ domain in Dicer has a similar function to its counterpart in Argonaute. Indeed, Dicer cleaves with increased efficiency if dsRNAs are first pre-treated with an RNase III enzyme that leaves two-nucleotide 30 overhangs ([5] J Silva and GJ Hannon, personal communication). In addition, miRNA precursors that are produced by Drosha, another RNase III enzyme that leaves 30 overhangs, are in turn substrates for Dicer. Therefore, specific recognition of 30 overhangs might be crucial for entry into the RNAi pathway. These observations led to models in which the PAZ domains of Argonaute in RISC and of Dicer specifically recognize siRNA 30 ends [4,6,7]. However, the sequence alignment of the PAZ domains from Argonaute and Dicer shows that there is a 20–30 Current Opinion in Structural Biology 2006, 16:5–11

amino acid insertion between b6 and b7 in the Dicer PAZ domain. This insertion is at the bottom of the RNA binding cleft, which suggests that the RNA in the PAZ domain of Dicer might be bound somewhat differently.

Molecular basis of siRNA recognition by the PAZ domain Structures of the PAZ domain in complex with nucleic acids revealed the mode of interaction and the details of siRNA recognition by the PAZ domain [9,10]. Structures of the nucleic acid bound forms are almost identical to those of the free PAZs, which indicates that the nucleic acid binding surface is pre-formed. Surprisingly, in the cocrystal structure of PAZ domain from human Argonaute1 (hAgo1) complexed with a 9-mer RNA oligonucleotide, the RNA formed an siRNA-like duplex — a pseudo-siRNA — that had a two-nucleotide overhang at each of the 30 ends even though there were three mismatches in the duplex [10]. The duplex portion of the RNA lies on the positively charged surface formed by the C-terminal tail of the PAZ domain and the top of its bbarrel (Figure 1). The two-nucleotide 30 -overhang of the pseudo-siRNA then makes a sharp turn, is inserted into the intersubdomain cleft, and interacts with residues previously identified by mutagenesis and nuclear magnetic resonance chemical shift perturbations (discussed above). The way in which the overhang is recognized by the PAZ domain displays several unexpected features (Figure 1). One would expect to see stacking interactions between aromatic residues and the bases of a single-stranded nucleic acid, as is the case for other single-stranded nucleic acid binding proteins [12]. However, the invariant aromatic residues in the cleft (Tyr309, Tyr314, His269 and Tyr277) make hydrogen bonds with the non-bridging oxygens of the phosphate between the two bases in the overhang, one of which is water-mediated. There is one base-stacking interaction, however, between the last base of the pseudo-siRNA and Phe292, securing this base into position. The hydroxyl group at the 30 end of the pseudosiRNA, another characteristic feature of an siRNA, is accommodated primarily by steric exclusion at the inside of the interdomain cleft although there is a hydrogen bond with a carbonyl group of Tyr336 that also helps to hold it in place. The 20 hydroxyl is anchored in place by backbone interactions to the amide of the same residue and to the carbonyl of His334. All of these interactions are also seen in the oligonucleotide-bound solution structure of the PAZ domain [9]. Curiously, the PAZ domain interacts exclusively with the strand of the siRNA duplex that has its 30 end inserted into the binding pocket. It does not appear to examine the 20 hydroxyl groups of sugar rings of RNA to discriminate RNA from DNA. However, several positively charged residues on the surface formed by the strand b2, b3 and www.sciencedirect.com

Argonaute and RNA Song and Joshua-Tor

Figure 1

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Figure 2

The structure of P. furiosus Argonaute. Ribbon representation of Argonaute that shows the N-terminal domain (blue), the ‘stalk’ (light blue), the PAZ domain (red), the Mid domain (green), the PIWI domain (purple) and the interdomain connector (yellow).

The structure of the PAZ domain of hAgo1 bound to a ‘mini-siRNA’. The PAZ domain is shown in grey in ribbon representation and the RNA is shown as a stick model with one strand drawn in purple and the other in cyan. The 30 end of the purple strand is bound in the cleft of the PAZ domain. The 30 end of the strand drawn in light blue is bound to another PAZ domain in the same manner in this crystal structure, but this second PAZ domain is not shown for clarity.

the b6–b7 loop interact with non-bridging oxygens of the backbone phosphates of one strand of the pseudo–siRNA duplex. Therefore, RNA preference might be determined through recognition of the characteristic spacing between the backbone phosphates, which is quite different between A-form RNA and B-DNA.

Argonaute: the catalytic engine of RNAi Argonaute is a major protein component of RISC — the RNAi effector complex. Together with an siRNA or miRNA it defines RISC. However, its function remained unknown until the crystal structure of a full-length Argonaute from the archaea Pyrococcus furiosus (PfAgo) was solved. This structure revealed that Argonaute is in fact the catalytic engine of RNAi, the endonuclease nicknamed Slicer, which cuts mRNA targets guided by siRNAs [13]. Argonaute consists of four domains. The N-terminal, middle and PIWI domains form a crescent-shaped base, with the PIWI domain at the center of the base (Figure 2). The PAZ domain is located above the base and is held by www.sciencedirect.com

a stalk-like linker region between the N-terminal and the PAZ domains. An interdomain connector cradles the structure. This architecture forms a large positively charged groove between the PAZ domain and the crescent base, and a smaller one between the N-terminal and PIWI domains. The PAZ domain of PfAgo is similar in structure to the human and fly Argonaute PAZ domains described above, except PfAgo PAZ has two a-helices in the small subdomain rather than a b-hairpin and an a-helix. The cleft of the PAZ domain, involved in binding to the twonucleotide overhang at the 30 end of the siRNA, faces one end of the primary groove of Argonaute. The primary sequence of PfAgo PAZ domain is poorly conserved compared with other PAZ domains. Despite the low sequence conservation, all residues involved in the recognition of the 30 two-nucleotide overhang of siRNAs fall in the same three-dimensional (3D) position as their counterparts in the other PAZs. Y212, Y216, H217 and Y190 of PfAgo are equivalent to residues Y309, Y314, H269 and Y277 of hAgo1. All are involved in making contact with the oxygens of the last phosphate of the 30 end of an siRNA. Interestingly, H217 of PfAgo and H269 of hAgo occupy the same 3D position although they are from entirely different parts of the backbone. Thus, the PAZ domain of PfAgo maintains the same structure as the other PAZ domains. In addition, crosslinking studies showed that PAZ binds the 30 end of an siRNA in the full length PfAgo (J-J Song and L Joshua-Tor, unpublished) and therefore it appears to have the same function as well. Current Opinion in Structural Biology 2006, 16:5–11

8 Protein–nucleic acid interactions

Figure 3

The active site of Argonaute and RNase H. (a) The active site in the PIWI domain of PfAgo showing a Mn2+ metal ion (purple) bound to the active site residues. The DDH motif is drawn in stick representation. Water molecules bound to the metal are shown in green. The metal ion here is equivalent to metal A in (b). (b) The active site of the D132 mutant of Bacillus halodurans RNase H in complex with the RNA–DNA hybrid. The active site residues and the RNA substrate are shown in stick representation, the divalent metal ions are drawn as purple, and the water molecules as green spheres. Carbons are drawn in grey (light for the protein and dark for the RNA), oxygens in red, nitrogens in blue and phosphorous in yellow. The two aspartates in the active site of Argonaute, Asp558 and Asp628, are equivalent to Asp71 and Asp132 in RNase H. Argonaute His745 is equivalent to Asp192 of RNase H. A second metal ion would probably bind to Argonaute in a substrate-bound form.

The Mid domain, at one end of the crescent, is similar to the sugar-binding domain of Lac-repressor, which binds lactose between two such domains. The PIWI domain, at the C-terminus of Argonaute, is located across the primary groove from the PAZ domain. It appears to reside only in Argonaute or Piwi proteins and when the first studies were performed not much was known about it from its sequence alone. It is thus the most surprising part of the Argonaute structure. The PIWI domain core fold clearly belongs to the RNase H family of enzymes, containing two highly conserved aspartates on adjacent b-strands. In fact, divalent metal ion binding showed that the PIWI domain in Argonaute contains three conserved catalytic residues composed of the two aspartates and a histidine, called the ‘DDH’ motif, which is analogous to the ‘DDE’ catalytic motif of RNase H fold enzymes [14]. Mutation of any of the equivalent DDH residues of human Argonaute2 [15,16] eliminates Slicer activity. This finding strongly implicated Argonaute to be the long-sought catalytic enzyme of RISC, dubbed ‘Slicer’, which slices the mRNA substrate guided by the siRNA. Moreover, the dependence of RISC Slicer activity on the presence of Mg2+ ions and the generation of similar products to RNase H family enzymes further underscores this conclusion. Recently, a crystal structure of a bacterial RNase H in complex with its double-stranded DNA–RNA substrate has shed light regarding this family of enzymes [17]. The two metal ion mechanism derived from this structure is Current Opinion in Structural Biology 2006, 16:5–11

shared with other members of the family, including transposases, intergrases and, most probably, the Argonautes. Indeed, one of these metal ions is equivalent to the one found in PfAgo and is coordinated by one of the conserved aspartates and another aspartate that is equivalent in position to the histidine of Argonaute (Figure 3). A second metal ion is coordinated by the first aspartate, the second conserved aspartate and a glutamate. Both metal ions are also coordinated by the substrate. The absence of a second metal ion in PfAgo is probable owing to its lower stability in the absence of substrate, as was the case for RNase H. In addition, RNase H uses four conserved carboxylates, whereas the transposases and integrases use three. A fourth conserved residue, a glutamate, was originally identified as a possible active site residue in Argonaute [13], but it appears to be a bit distant from the metal and its mutation to alanine did not affect activity [16]. The genome of another archaea, Archaeoglobus fulgidus, encodes a two-domain protein that contains the Mid and PIWI domains alone, also termed the A and B domains. The crystal structure of this PIWI-domain protein, AfPiwi (not to be confused with the Piwi proteins mentioned above, which do contain PAZ domains), shows these two domains to have an essentially identical structure to those in PfAgo, although they are slightly rotated relative to each other [18]. There is an additional 40residue N-terminal region that sits across the interface between these domains. As this protein does not contain the catalytic aspartates, it does not bind a divalent metal ion in the active site; however, a Cd2+ ion is bound to the www.sciencedirect.com

Argonaute and RNA Song and Joshua-Tor

C-terminal carboxylate of the protein at a conserved region in the protein. The C terminus of both AfPiwi and PfAgo lies at the interface between the Mid and Piwi domains.

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Figure 4

More recently, the structure of a eubacterial Argonaute protein from Aquifex aeolicus (AaAgo) was determined and was shown to have identical domain architecture to PfAgo [19]. However, the relative orientation of the PAZ domain is quite different. There is a Ca2+ ion in the active site and in this case there are three aspartates, two that correspond to the active site aspartates and one that corresponds to the histidine in PfAgo. Interestingly, this protein was shown to have RNase H-like activity, whereby it shows preference for a DNA–RNA hybrid as a substrate. It works better at higher temperatures, for example 55 8C, and prefers to use Mn2+ rather than Mg2+ as the divalent cation.

RNA binding As mentioned previously, Argonaute displays a prominent positively charged groove along the protein, which is presumed to be the RNA-binding groove [13]. The opening of the PAZ cleft, shown to bind the 30 end of the siRNA, is on one side of the groove and the catalytic residues of the PIWI domain are in its center. On the basis of known biochemical properties of RISC cleavage and the structures of PAZ domains complexed with nucleic acids, a model for siRNA and target binding was proposed [13]. In this model, the PAZ domain grips the 30 end of the ss-siRNA, and the siRNA–mRNA duplex sits along the positively charged groove (Figure 4). The 50 region of the mRNA lies between the PAZ and N-terminal domains. Importantly, the catalytic DDH motif of the PIWI domain then faces the scissile bond of a target mRNA substrate opposite nucleotides 10 and 11 from the 50 end of the siRNA, consistent with the mRNA slicing position known from previous biochemical studies. From crosslinking studies, it was observed that both hAgos 1 and 2 recognize the 50 end of the siRNA guide [16]. Recently two crystal structures of AfPiwi in complex with ds-siRNA-like oligonucleotides were determined; these address the 50 end recognition of the guide siRNA by the Argonautes [20,21]. 50 end recognition is an important feature of RISC. Slicing position is determined from the 50 end of the siRNA, which is characterized by the presence of a phosphate at that end as a result of Drosha and Dicer processing. In both crystal structures, the 50 nucleotide of the strand denoted as the siRNA is unpaired from its counterpart on the other strand and sits in a cleft previously suggested to be the 50 -end binding region [18] (Figure 5). The 50 phosphate is anchored to the cleft through interactions with a divalent metal ion that is, in turn, bound to the C-terminal carboxylate at the interface between the Mid and PIWI domains. The metal ion also coordinates the third phosphate of the same strand. Also contacting the 50 phosphate are Tyr123, Lys127, Gln137 www.sciencedirect.com

A model for siRNA-guided mRNA cleavage by Argonaute. The 30 end of the siRNA, shown in purple, was placed by superposition of the hAgo1 PAZ domain–RNA complex on the PfAgo PAZ domain. The 50 end of the mRNA substrate, shown in light blue, was modeled by using the passenger strand (the ‘passenger strand’ is the strand hybridized to the guide strand in an siRNA, sometimes referred to as the ‘sense strand’) from the same superposition and extending it by two nucleotides towards its 50 side to lie between the N-terminal and PAZ domains, and towards its 30 side along the positively charged major groove of PfAgo towards the PIWI domain active site residues shown in red. The phosphate between nucleotides 11 and 12 from the 50 end of the mRNA falls near the active site residues.

and Lys163, all of which are well-conserved residues. The aromatic ring of Tyr123 stacks on the 50 base of the guide. A helix from the Mid domain appears to separate the two strands at the 50 end. This unpairing of the first putative guide-target ‘basepair’ is in agreement with the observation that base pairing at this position does not appear to be required for target recognition [22]. As expected, most of the interactions observed are sequence independent. Another suggestion for the location of the 50 -phosphate, near a region termed the ‘PIWI box’, was made based upon a study in which PfAgo crystals were soaked with tungstate, a moiety that might substitute for phosphate [16]. This location is close to the corresponding position of the 30 region of target strand in the AfPiwi–RNA observed in one of the two structures [21]. It should be noted that the metal ion bound to the C terminus observed in both RNA-bound and free forms of AfPiwi is not observed in PfAgo, even when crystals were soaked with Mn2+ [16]. It is possible that this metal ion would be stabilized by binding of the guide strand if binding is similar to binding in AfPiwi. The positioning of the RNA is slightly different in the two AfPiwi–RNA complexes Current Opinion in Structural Biology 2006, 16:5–11

10 Protein–nucleic acid interactions

Figure 5

kinetics for this minimal enzyme are remarkably similar to those determined for RISC from either flies or human cell extracts, indicating that other factors are not needed to assist in binding and cleavage. However, an ATP dependence on turnover, characteristic of more complete RISC complexes, is absent from this minimal form, indicating that another protein assists in product release using ATP. Such a factor might trigger domain movement in Argonaute, a feature that is intimated from the various structures described above.

Specializing Agos

The structure of AfPiwi in complex with RNA. AfPiwi is shown in a semi-transparent ribbon representation with the Mid (also called the A) domain in green and the PIWI (also called the B) domain in dark purple. RNA is shown in stick representation with the strand representing the siRNA shown in purple and the strand representing the substrate in light blue. The divalent metal ion bound to the C-terminal carboxylate of the protein and the 50 phosphate as well as the phosphate of the third nucleotide is shown as a dark pink sphere.

after the first base pair, although the protein does not appear to make contacts to the target strand past this position. Contact to the target strand as well as some repositioning of the duplex might change in the context of a full-length Argonaute protein, which has a more restricted binding groove for the RNA than the rather open PIWI-domain protein. We also expect some domain movement of the protein and adjustments of RNA positioning to occur to avoid clashes with other parts of the full-length protein and to bring the target RNA in the correct position for cleavage. Domain movement would probably occur upon binding of the guide strand and more so upon binding of the target strand, perhaps during catalytic turnover of the slicing Argonautes. Study of a complex of a full-length Argonaute with RNA would no doubt address many of these questions.

Minimal RISC The expression of hAgo2 in Escherichia coli provided an important opportunity to examine which features of RISC activity reside in Argonaute itself and which must reside elsewhere [16]. First, we learned that hAgo2 and an siRNA guide are all that are needed to carry out slicing. The 50 phosphate of the siRNA is important for enzyme stability as well as for RISC fidelity. Single turnover Current Opinion in Structural Biology 2006, 16:5–11

Not all Argonautes are capable of slicing mRNAs. In humans only one of the four Argonaute subfamily proteins, hAgo2, possesses slicing activity. Of course, the presence of the crucial active site residues that have been identified to date is a prerequisite for a catalytically competent active site. However, this does not appear to be the whole story. The histidine is absent in hAgo1, and in hAgo4 one of the aspartates is missing; however, hAgo3 has all three and is still inactive for slicing [15,16]. In Arabidopsis there are ten Argonautes. Eight of these have an intact DDH motif, but the other two, Ago2 and 3, have an aspartate at this position, which in analogy to RNase H should be able to substitute for the histidine. AtAgo1 was shown to be an RNA Slicer [23] but it is not clear whether the others are RNA Slicers or whether this activity is required for their function. AtAgo4, for example, appears to be involved in chromatin silencing, however, we do not know if it uses slicing activity in this role. In flies, ago1 mutants are defective in miRNA-mediated silencing, but not in siRNA-directed cleavage, whereas ago2 mutants are defective in siRNAdirected cleavage but not in miRNA silencing [24]. Caenorhabditis elegans have as many as 27 Argonaute proteins, and although there appears to be some specialization their roles are still being discovered [25]. On the other extreme, Schizosaccharomyces pombe has only one Argonaute (Ago1), which is involved in both RNAi and transcriptional silencing [26], yet it is unclear at this time whether its slicing activity is required for the latter. Is the diversification of the Argonautes a consequence of the siRNA or miRNA that they recruit? Is the conformation of the protein dependent on the substrate it targets, or does the protein conformation dictate its targets? For the four human Argonautes, the distribution of miRNAs appears to be identical across them all when they are ectopically expressed [15]. Perhaps other proteins that interact with the various Argonautes are responsible for modulating their activity and function.

Acknowledgements We thank Michelle A Carmell and Niraj H Tolia for their critical comments. This work was supported by a grant from the National Institutes of Health (GM072659 to LJ). www.sciencedirect.com

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10. Ma JB, Ye K, Patel DJ: Structural basis for overhang-specific  small interfering RNA recognition by the PAZ domain. Nature 2004, 429:318-322. The crystal structure of hAgo1 PAZ domain in complex with a ‘minisiRNA’ showing the details of the interactions between the PAZ domain and the RNA, in particular the interactions with the 3’-overhang. 11. Theobald DL, Mitton-Fry RM, Wuttke DS: Nucleic acid recognition by OB-fold proteins. Annu Rev Biophys Biomol Struct 2003, 32:115-133. 12. Antson AA: Single-stranded-RNA binding proteins. Curr Opin Struct Biol 2000, 10:87-94. 13. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L: Crystal structure  of Argonaute and its implications for RISC slicer activity. Science 2004, 305:1434-1437. The first crystal structure of a full-length Argonaute reveals its function as ‘Slicer’ — the catalytic component of RISC. 14. Yang W, Steitz TA: Recombining the structures of HIV integrase, RuvC and RNase H. Structure 1995, 3:131-134.

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15. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ,  Hammond SM, Joshua-Tor L, Hannon GJ: Argonaute2 is the catalytic engine of mammalian RNAi. Science 2004, 305:1437-1441. This study shows that of the four human Argonautes, only Argonaute2 functions as Slicer. 16. Rivas FV, Tolia NH, Song JJ, Aragon JP, Liu J, Hannon GJ,  Joshua-Tor L: Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol 2005, 12:340-349. The presence of human Argonaute2 with an siRNA is sufficient for slicer activity. This system is used to show which features of RISC activities reside in Argonaute and which must reside in a different component of RISC. The active site of Argonaute is shown here to have a ‘DDH’ motif. 17. Nowotny M, Gaidamakov SA, Crouch RJ, Yang W: Crystal  structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 2005, 121:1005-1016. The first RNase H structure in complex with its substrate provides important insights that are relevant for Argonaute substrate recognition and slicing activity. 18. Parker JS, Roe SM, Barford D: Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J 2004, 23:4727-4737. 19. Yuan YR, Pei Y, Ma JB, Kuryavyi V, Zhadina M, Meister G,  Chen HY, Dauter Z, Tuschl T, Patel DJ: Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol Cell 2005, 19:405-419. Crystal structure of another archaeal full-length Argonaute protein showing domain movement of the PAZ domain. It also shows it to have preference for DNA–RNA hybrid substrates. 20. Ma JB, Yuan YR, Meister G, Pei Y, Tuschl T, Patel DJ: Structural  basis for 5(-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 2005, 434:666-670. This reference and [21] describe two crystal structures of a Piwi-domain protein in complex with RNA, and implications for 50 -end siRNA recognition. 21. Parker JS, Roe SM, Barford D: Structural insights into mRNA  recognition from a PIWI domain-siRNA guide complex. Nature 2005, 434:663-666. For annotation, see [20]. 22. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS: MicroRNA targets in Drosophila. Genome Biol 2003, 5:R1. 23. Baumberger N, Baulcombe DC: Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 2005, 102:11928-11933. 24. Okamura K, Ishizuka A, Siomi H, Siomi MC: Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev 2004, 18:1655-1666. 25. Kim JK, Gabel HW, Kamath RS, Tewari M, Pasquinelli A, Rual JF, Kennedy S, Dybbs M, Bertin N, Kaplan JM et al.: Functional genomic analysis of RNA interference in C. elegans. Science 2005, 308:1164-1167. 26. Sigova A, Rhind N, Zamore PD: A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. Genes Dev 2004, 18:2359-2367.

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