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Curr Opin Struct Biol. Author manuscript; available in PMC 2010 Aug 1.
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PMCID: PMC2855239

Structural Insights into RNA Interference


Virtually all animals and plants utilize small RNA molecules to control protein expression during different developmental stages and in response to viral infection. Structural and mechanistic studies have begun to illuminate three fundamental aspects of these pathways: small RNA biogenesis, formation of RNA-induced silencing complexes (RISCs) and targeting of complementary mRNAs. Here we review exciting recent progress in understanding how regulatory RNAs are produced and how they trigger specific destruction of mRNAs during RNA interference (RNAi).


Small RNA molecules play central roles in regulating eukaryotic gene expression by means of RNA interference (RNAi) and related pathways [1],[2]. Fundamentally, RNA-mediated genetic control begins with the production of ~20-30-nucleotide RNAs whose sequences can base pair with segments of mRNA transcripts. Once generated, these microRNAs (miRNAs) or small interfering RNAs (siRNAs) assemble into large multi-protein effectors, called RNA-induced silencing complexes (RISCs), which bind to target transcripts and trigger their destruction.

The production of miRNAs and siRNAs requires endonucleolytic processing of double-stranded precursors. Transcribed initially as long stem-loop structures in the nucleus, miRNAs are recognized by the protein DGCR8, which recruits the RNA to the RNase III-family endonuclease Drosha for initial double-strand cleavage (Fig. 1a). The resulting pre-miRNA is exported to the cytoplasm for cleavage into the mature miRNA duplex by a second RNase III-family endonuclease called Dicer (Fig. 1b). SiRNA production results from Dicer-mediated cleavage of long double-stranded RNAs that are either generated in the cell or introduced by viruses or transfection (Fig. 1c). In either case, the resulting duplex includes both the antisense guide strand and the sense, or passenger, strand (Fig. 1d).

Figure 1
Gene silencing by small RNAs

Two features of miRNAs and siRNAs ensure their efficient incorporation into the RISC complex: a specific length of the RNA duplex, and characteristic 5′ and 3′ ends carrying a phosphate group and a dinucleotide overhang, respectively. Structural studies of a prokaryotic RNase III and a eukaryotic Dicer enzyme have provided insights into how these critical features of siRNAs and miRNAs are generated [3-7].

MiRNAs and siRNAs subsequently associate with members of the Argonaute (Ago) family of proteins, which function as the core components of RISCs (Fig. 1e). A key aspect of this process is the loading of a guide strand into Ago, and the dissociation and/or destruction of the passenger strand. RISCs use the small RNAs as guides for sequence-specific silencing of complementary target RNA molecules by repressing their translation (Fig. 1f) or inducing their degradation (Fig. 1g). A specialized nuclear Argonaute-containing complex, known as the RNA-induced transcriptional silencing (RITS) complex, mediates transcriptional gene silencing by inducing heterochromatin formation in certain organisms [8]. Proteins belonging to the Argonaute family are also involved in the biogenesis of a third class of small RNAs, the Piwi-interacting RNAs (piRNAs) [9].

Key to the activities outlined above is the ability to specifically recognize, process and traffic small RNAs and their target mRNAs. Here we highlight recent structural and mechanistic insights into small RNA biogenesis, assembly with RISC and target recognition.

RNA manipulation by dsRBPs during biogenesis

Small RNA precursors contain distinctive characteristics that contribute to their recognition by the biogenesis machinery. In some cases, domains within partner proteins are uniquely tailored to recognize these features. For example, the PAZ domain found within Dicer and Ago specifically binds the 3′ overhangs of Drosha and Dicer products. In contrast, several silencing factors utilize more generic dsRNA binding domains (dsRBDs) to recognize the double stranded elements within precursor RNAs. Both Drosha and Dicer associate with dsRBD-containing proteins (dsRBPs), which are critical for the specific binding and cleavage of their substrates. These RNA-binding cofactors generally contain more than one dsRBD, providing a surprising diversity of lengths of RNAs bound by various Dicer-associated dsRBPs. For example, the Caenorhabditis elegans dsRBP RDE-4 binds RNA cooperatively, affording it an increased affinity for the longer siRNA precursors targeted by its associated Dicer [10]. In contrast, human TRBP does not display cooperative dsRNA binding, and instead binds RNAs of various lengths with similar affinities [10]. TRBP plays an important role in transferring mature siRNA duplexes to RISC [11-13], so must be able to bind short RNAs in addition to longer substrates.

While dsRBD-RNA interactions have been a popular target of structural studies, structures of multiple dsRBD-containing proteins are rare. The recent crystal structure of the dsRBD region of human DGCR8, the RNA-binding partner of Drosha, has provided important insights into the mechanism of substrate recognition and product length determination by a protein containing two dsRBDs [14]. Individual dsRBDs adopt αβββα folds, forming a compact α/β sandwich that contacts both the major and minor grooves on one face of the RNA [15]. In the crystal structure of DGCR8, the two dsRBDs form a pseudo two-fold symmetry based mainly on mutual interactions with a C-terminal α-helix (Fig. 2a). The α-helices and loop that contact RNA lie on opposite sides of the molecule, suggesting that a single RNA helix bound by DGCR8 would be substantially kinked by interactions with the two dsRBDs (Fig. (Fig.1a,1a, ,2a).2a). Intriguingly, the approximate length of an RNA that could bind in this conformation is 33 base pairs, similar to the length of a typical pri-miRNA hairpin, suggesting a mechanism by which DGCR8 identifies the correct substrates for Drosha.

Figure 2
Structures of two dsRBPs involved in small RNA biogenesis

Although the structure of DGCR8 provides preliminary insight into how multiple dsRBDs within a single protein may cooperatively bind an RNA target, several questions still remain. Biochemical evidence suggests that DGCR8 binds to the base of pri-miRNA hairpins, an interaction that is facilitated by association with flanking single-stranded sequence on either side of the RNA stem. Cleavage occurs ~11 base-pairs into the hairpin [15], suggesting that DGCR8 positions Drosha at an appropriate distance from the base of the stem (Fig. 1a). This mechanism for generating products of a specific length is reminiscent of Dicer, in which the distance between the 3′-overhang-binding PAZ domain and the catalytic center of the enzyme determines the length of the product (Fig. 1b, c) [5]. However, for the Drosha-DGCR8 complex, the structural basis for product length definition remains unclear. Structures of DGCR8 bound to RNA or Drosha would greatly increase our understanding of how the dsRBP recognizes the ssRNA junction at the base of the hairpin, while concurrently positioning Drosha for cleavage.

In plants, an additional processing step occurs following Dicer cleavage, in which the 2′-hydroxyl on the 3′ end of the siRNA duplex is methylated by the methyltransferase HEN1. In Arabidopsis thaliana, HEN1 displays a preference for 21-24 nucleotide duplexes with 2 nucleotide overhangs, suggesting that RNA recognition may proceed through mechanisms similar to those observed for other components of the biogenesis machinery [16]. Indeed, the recent crystal structure of HEN1 elegantly condenses several of the themes of small RNA recognition discussed above into a single molecule (Fig. 2b) [17]. In the structure, HEN1 is bound to a 22-nucleotide RNA duplex, an interaction that is mainly mediated by two dsRBDs that simultaneously bind the same stretch of RNA on opposite faces (Fig. 2b). This arrangement is in sharp contrast to the dsRBDs of DGCR8, where the rigid orientation of the domains restricts the protein from wrapping around the RNA, allowing the protein to bind RNAs of a specific length along only one side of the duplex. The HEN1 dsRBDs span only 16-base pairs of the helix, requiring that the final few base pairs on either end interact with other domains to determine substrate length.

One domain that restricts substrate length is the catalytic methyltransferase domain, which caps the RNA and interacts with an elongated loop within the first dsRBD. The 3′ nucleotide in the active site is mainly oriented through coordination of the 2′ and 3′ hydroxyls by a Mg2+ ion that is bound by four conserved residues, as well as stacking interactions with the ribose group of the cofactor adenosyl-L-homocysteine (AdoHcy) (Fig. 2c). The penultimate nucleotide of the modified strand flips out of the A-form helix, making specific contacts with two residues distant from the active site. This conformation allows the 3′ nucleotide to stack on the final base pair of the helix, likely providing stability for substrates with 2 nucleotide overhangs (Fig. 2c).

On the other end of the RNA, a La motif completes the substrate length determination. La protein, from which the La motif originates, recognizes the 3′ ends of nascent RNA Polymerase III transcripts, forming specific contacts between its N-terminal domain (NTD) and the terminal 3 nucleotides of the transcript [18]. However, the RNA interactions observed for the HEN1 La motif differs significantly from the RNA-NTD interaction. The HEN1 La motif replaces the 3′ binding pocket of the NTD with two conserved loops that form a platform on which the 5′ end of the modified strand stacks (Fig. 2d), an interaction that is essential for substrate binding and methylation. This variation in specific RNA contacts between the La-NTD and the HEN1 La motif presents an intriguing example of how a single RNA binding motif can be modified slightly to provide starkly different binding environments. The 2-nucleotide overhang of the unmodified strand is also bound by the La motif (Fig. 2d); however, this contact is not essential, consistent with biochemical evidence that the length of the unmodified overhang is not important for HEN1 activity [16]. Overall the HEN1 structure is an excellent example of how RNA silencing factors utilize a large variety of motifs to specify substrate length, and provides an interesting counterpoint to the molecular ruler observed for Dicer product length determination [5].

RISC Loading

Following Dicer cleavage, mature miRNA or siRNA duplexes must be transferred from Dicer to Argonaute to form the active RISC. This process leads to the selection of the guide strand and removal of the passenger strand. In humans, the minimal components required for loading Argonaute 2 (Ago2) are Dicer complexed with its dsRBP cofactor, TRBP, and a mature siRNA duplex [13]. The human RISC loading complex (RLC) and its components have recently been analyzed by negative-stain electron microscopy (Fig. 3a, b), providing a structural framework for testing how siRNA duplexes may be passed from Dicer-TRBP to Ago2 during RISC loading [19, 20].

Figure 3
Cryo-EM reconstructions of the human RISC loading complex

The EM reconstruction of human Dicer reveals an L-shaped molecule containing a central cleft within the long axis (Fig. 3a) [19, 20]. The short arm of the L corresponds to the N-terminal helicase (DE×H/D) domain, based on analysis of a deletion mutant missing the ~80 kD helicase region [19]. The crystal structure of Giardia intestinalis Dicer, which lacks the helicase domain, can be modeled into the Dicer reconstruction in two different orientations, with the RNase III domains positioned toward either the base or the top of the L [5, 20]. However, given the location of the N-terminal helicase domain at the base of the L, the latter orientation seems more likely, as the C-terminal RNase III domains are distant from the N-terminus in the G. intestinalis Dicer structure (Fig. 3c).

EM reconstructions of various combinations of RLC components were used to dissect the remaining make-up of the full RLC reconstruction [19]. These analyses suggest that TRBP interacts flexibly with the helicase domain of Dicer, while Ago2 stretches between TRBP and the long arm of Dicer, forming a closed complex in some particles (Fig. 3b). The Ago2-Dicer interaction is thought to occur between the catalytic PIWI domain of Ago2 and the RNase IIIa domain of Dicer, based on previous biochemical studies [21]. This information, coupled with the positioning of G. intestinalis Dicer, suggests the location of Ago2 within the EM reconstruction of Dicer plus Ago2, allowing for the modeling of the Thermus thermophilus Ago crystal structure [22] into this reconstruction (Fig. 3b, c).

In order to determine how an siRNA duplex could be accommodated within the RLC, a 22 base-pair siRNA was modeled along the putative RNA-binding face of Dicer, revealing that the dsRNA fits perfectly within the gap between Ago2 and Dicer (Fig. 3c). In an alternative RNA-binding model, the 22 base-pair duplex can also be stretched between the PAZ domains of Ago2 and Dicer, suggesting that the two domains could bind both ends of the siRNA simultaneously (Fig. 3d). This observation suggests an intriguing possibility for an intermediate RLC conformation during RNA hand-off. Although TRBP is known to be important for recruiting siRNAs to Ago2 [11], its exact role in this process is unknown. The location and flexibility of TRBP suggested by the EM model may allow the dsRBP access to the PAZ domain of Ago2. TRBP could therefore bridge the gap between Dicer and Ago2, retaining the siRNA within the RLC once Dicer releases it, and positioning the correct end of the siRNA for binding by the Ago2 PAZ domain. The process of orienting the siRNA during transfer to Ago2 is particularly important for strand selection, which is based primarily on thermodynamic asymmetry of the small RNA duplex. TRBP may have an additional proofreading role for strand selectivity during RISC loading by sensing the relative thermodynamic stability of either end of the duplex.

The final step of RISC activation, passenger strand removal, has recently been the subject of several biochemical studies. In Drosophila melanogaster, passenger strand removal proceeds through different mechanisms depending on which Argonaute homolog is present in RISC. Upon transfer of duplex siRNAs to RISC, Drosophila Ago2 cleaves the passenger strand [23-25] and the endonuclease C3PO removes the cleavage products, activating the Ago2-guide strand complex [26]. In contrast, Ago1, which binds miRNAs and has relatively weak cleavage activity compared to Ago2 [27], unwinds the miRNA duplex through a slicer-independent mechanism [28]. Human Ago2-containing RISC likely follows the slicer-dependent mechanism for passenger strand removal [29], although human C3PO has not been confirmed as a RISC activator. Future biochemical and structural studies of the minimal human RLC associated with its other binding partners may shed more light on the activation of Ago2-RISC.

Mechanisms of RNA silencing

Following RISC loading, the Ago-guide strand complex binds its target mRNA, which occurs through base pairing of the guide strand with sequences within the 3′-untranslated region (UTR) of the mRNA. Full guide-target strand complementarity is required for target strand cleavage through the endonucleolytic “slicer” activity of Ago [30], a common mechanism for RNA silencing by siRNAs. Ago family proteins contain two distinctive domains: the PAZ domain, which binds the 3′-end of the guide strand, and the RNase H-like PIWI domain, which houses the slicing active site (Fig. 4a). A subset of Ago proteins also contain a MID domain, which forms a binding pocket for the 5′-phosphate of the guide strand through extensive hydrogen bonding with conserved residues and a Mg2+ ion. Several structures of prokaryotic Ago homologs have revealed the arrangement of these domains [31-38], and a recent set of structures of Thermus thermophilus Ago bound to several combinations of guide and target strands have provided insight into the mechanisms of substrate selection and slicing catalysis [22, 39, 40].

Figure 4
Structures of full length Argonaute from Thermus thermophilus bound to guide DNA and target RNA

In the structure of T. thermophilus Ago bound to a 21-nucleotide guide strand DNA [40], the guide strand stretches between the PAZ and MID/PIWI domains, with nucleotides 2-10 adopting a stacked, partially helical conformation that is facilitated by an extensive hydrogen-bonding network between the phosphate backbone of nucleotides 2-6 and residues in the MID and PIWI domains. Nucleotides 12-17 are mostly disordered in the structure, suggesting that they do not strongly contact the protein, while 18-21 are stacked and anchored by backbone interactions with residues in the PAZ binding pocket. This arrangement presents the seed sequence, located between nucleotide 2-8, with its Watson-Crick face exposed to solvent, consistent with the importance of this region in target recognition [41]. Surprisingly, the guide strand is distorted between nucleotides 10 and 11, where the DNA forms interactions with two highly conserved arginine residues (Fig. 4b). This conformation suggests that the guide-Argonaute complex must undergo a significant structural change upon binding of the target strand, resulting in an uninterrupted helix at the cleavage site.

Recent structural studies of a catalytically inactive Argonaute complexed with a fully complementary guide DNA strand and target RNAs of various length (Fig. 4a) offer the first detailed illustration of the slicer active site with bound substrate, while also providing insight into the conformational rearrangements that occur upon target binding [39]. The RNase H-like active site of T. thermophilus Argonaute contains three aspartate residues that contribute to catalysis. In the catalytic mutant structure of Argonaute, the scissile phosphate at the point of cleavage (10-11 step) in the target strand is adjacent to the three aspartate residues (Fig. 4c). Intermolecular hydrogen-bonds between the protein and the phosphate backbone of the target strand near the cleavage site stabilize the active site, but the target strand is mainly positioned through interactions with the guide strand. As observed for the binary guide-protein structure, the seed sequence of the guide strand is firmly held by the protein through a hydrogen-bonding network. Crystals grown in the presence of increasing amounts of Mg2+ contain one or two metal ions coordinated between the Asp residues and the cleavage site, underscoring the importance of divalent metal ions for slicing activity.

As predicted from the binary Argonaute-guide strand structure, a conformational change between nucleotides 10 and 11 of the guide strand is observed upon target binding, with the kinked arrangement giving way to a more canonical A-form helical geometry (Fig. 4b, c). This conformational change is accompanied by a shift in the overall structure of Argonaute, with the PAZ domain pivoting away from the MID/PIWI region to form a more “open” conformation (Fig. 4d). The opening of the protein structure allows the guide and target strand to zip up, as illustrated by structures of the ternary complex with target strands of increasing size. Nucleation of the guide-target helix leads to the eventual release of the guide 3′ end, as further movement of the PAZ domain becomes physically impossible (Fig. 4a); however, the N-terminal domain of the protein blocks base pairing beyond nucleotide 16 of the guide strand, indicating that the entire guide-target duplex never forms in the context of RISC. Taken together, these observations reveal a flexibility of Ago that allows it to facilitate target binding and form a catalytically competent active site.


Our understanding of the molecular mechanisms of RNA silencing has expanded tremendously over the last several years, due in large part to structural insights of RNAi-related proteins from lower organisms. High-resolution structures of silencing factors from plants and animals remain elusive and represent the next major step for structural biology of RNAi.


We thank D. Patel for communicating results in advance of publication and members of the Doudna laboratory for helpful discussions. Research in the Doudna laboratory is supported by the Howard Hughes Medical Institute and the National Institutes of Health. D.G.S. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation.


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