Dicer uses distinct modules for recognizing dsRNA termini

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Science  19 Jan 2018:
Vol. 359, Issue 6373, pp. 329-334
DOI: 10.1126/science.aaq0921

Substrate recognition by Dicer elucidated

The Dicer protein generates short RNAs from double-stranded RNA (dsRNA) substrates and is critical for RNA interference and antiviral defense. Sinha et al. report structures of a Drosophila Dicer protein that shed light on its two distinct mechanisms for recognizing and cleaving substrates: adenosine triphosphate (ATP)-independent, distributive cleavage of 3′-overhang dsRNAs and ATP-dependent, processive threading of blunt-end dsRNAs. This flexibility might provide invertebrates with the optimization capabilities needed for antiviral defense.

Science, this issue p. 329


Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3′ overhanging termini are cleaved distributively. To understand this discrimination, we used cryo–electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3′ overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate–dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers.

Dicer ribonucleases cleave double-stranded RNA (dsRNA) precursors to generate microRNAs (miRNAs) and small interfering RNAs (siRNAs) (1, 2). In concert with Argonautes, these small RNAs bind complementary mRNAs to down-regulate their expression. miRNAs are processed by Dicer from small hairpins, whereas siRNAs are typically processed from longer dsRNA, from endogenous sources (3), or from exogenous sources such as viral replication intermediates (46). Some organisms, such as Homo sapiens and Caenorhabditis elegans, encode one Dicer that generates miRNAs and siRNAs, but other organisms have multiple Dicers with specialized functions.

Dicers exist throughout eukaryotes, and a subset have an N-terminal helicase domain of the RIG-I-like receptor (RLR) subgroup (7) (Fig. 1A and fig. S1A). RLRs often function in innate immunity (8), and Dicer helicase domains sometimes show differences in activity that correlate with roles in immunity. For example, Drosophila melanogaster expresses two Dicers, one specialized for processing miRNAs (dmDcr-1) and a second for siRNAs (dmDcr-2) (9). dmDcr-1 has a degenerate helicase domain and is an adenosine triphosphate (ATP)–independent enzyme (10), whereas dmDcr-2, with dedicated antiviral roles (1113), has a conserved helicase domain that hydrolyzes ATP (1417). Under certain conditions, Homo sapiens Dicer-1 (hsDcr-1) also generates viral siRNAs (18, 19). However, despite conservation of its helicase domain, hsDcr-1 does not hydrolyze ATP in vitro (20), and its helicase domain is not implicated in viral siRNA biogenesis in vivo (19). Differences in activities of the helicase domain of vertebrate and invertebrate Dicers may reflect distinct roles in antiviral defense.

Fig. 1 Cryo-EM reconstructions of apo-dmDcr-2 and dmDcr-2•BLT dsRNA•ATP-γS.

(A) dmDcr-2 domains numbered at boundaries. Mutations/deletions are in bold and are designated in text as superscripts. All mutations were in the context of the full-length protein, unless specified by Δ. Two full-length variants had multiple mutations: PP (H743A, R752A, R759A, R943A, and R956A) and RIII (D1217A and D1476A). (B) Cryo-EM density map of apo-dmDcr-2 (7.1 Å) fitted with homology models of subdomains. RIIIa, RNase IIIa; RIIIb, RNase IIIb. (C) Homology model of apo-dmDcr-2 in open conformation, based on apo-RIG-I, and fitted as a rigid body into an 8.7-Å cryo-EM density map (see also figs. S4A and S5, B to D). (D) Cryo-EM reconstruction of dmDcr-2RIII•BLT dsRNA•ATP-γS showing helicase in closed, ligand-bound conformation. (E) Superimposition of open (light) and closed (dark) helicase conformations showing clamping of Hel2 and Hel2i on BLT dsRNA. Arrow, direction of clamping. (F) EM density and modeling of BLT single- and double-stranded RNA.

dmDcr-2 activity depends on termini of its dsRNA substrates (15, 16). Blunt (BLT) termini promote a processive reaction whereby multiple siRNAs are produced before dmDcr-2 dissociates, and this reaction requires a functional helicase domain and ATP (15, 17). In contrast, dsRNAs with 3′ overhanging (3′ovr) termini promote an ATP-independent, distributive cleavage, whereby dmDcr-2 dissociates after each cleavage. hsDcr-1 does not require ATP for processing BLT or 3′ovr termini (fig. S1B, C and D, lanes 5, 7, 10, and 12), suggesting that, at least in vitro, cleavage of BLT dsRNA is not dependent on its helicase domain.

To understand the mechanism of terminus discrimination by dmDcr-2, we used cryo–electron microscopy (cryo-EM) to determine structures of dmDcr-2 alone and in complex with a BLT 52 base pair (bp) dsRNA (52 dsRNA) and ATP-γS (Fig. 1, figs. S2 to S10, and table S1). We used full-length dmDcr-2 with a point mutation in each ribonuclease III (RNase III) domain to preclude dsRNA cleavage (dmDcr-2RIII) (Fig. 1A). ATP hydrolysis is required for processive cleavage of BLT dsRNA, and dmDcr-2 cannot hydrolyze ATP-γS, which stabilizes a helicase-dependent conformation of dmDcr-2 (16).

The structure of apo-dmDcr-2RIII (Fig. 1, B and C, and figs. S2 to S5) reiterated the “L shape” of lower resolution (~15 to 30 Å) EM reconstructions of hsDcr-1 (2123). Our 7.1-Å EM density map (Fig. 1B and fig. S2C) enabled fitting (fig. S4, B to F) and homology modeling (Fig. 1B) of Platform-PAZ domains at the cap and tandem RNase III domains in the core. An additional round of three-dimensional (3D) classification (fig. S3) revealed an 8.7-Å map (fig. S2C), allowing assignment of the helicase domain at the base (Fig. 1C and fig. S4A). Fitting of related apo-helicases into the EM density is consistent with the helicase domain adopting an open conformation (fig. S5, A to D).

The 2D class averages of the dmDcr-2 complex revealed protein with well-resolved secondary structure features bound to the BLT dsRNA terminus (fig. S6, B and C). Some protein density was missing, and because control experiments indicated that protein on the grid was intact (fig. S7A), this was likely due to inherent flexibility (fig. S7B). Measurements of the dsRNA, guided by major grooves, showed that visible protein footprinted ~8 to 9 bps (fig. S6B). The crystal structure of RIG-I’s helicase domain bound to dsRNA has a similar footprint (24, 25), suggesting that dmDcr-2’s helicase domain bound to BLT dsRNA termini. Indeed, our 6.8-Å reconstruction of the complex (figs. S6E and S8) resembled RIG-I in a closed conformation (Fig. 1D and fig. S9). The Hel1 and Hel2 subdomains of RIG-I’s helicase, along with the pincer helices (fig. S1A), could be fit as a single rigid body (fig. S9A). The reconstruction also revealed a helical bundle characteristic of the Hel2i subdomain that could be fitted separately as a rigid body (fig. S9B). These fittings enabled a homology model of dmDcr-2’s helicase domain bound to BLT dsRNA (Fig. 1D and fig. S9, D and E).

Our models of dmDcr-2’s helicase in open (apo) and closed (substrate-bound) conformations implied clamping of the helicase on BLT dsRNA termini (Fig. 1E and movies S1 and S2). In the open conformation, Hel2 and Hel2i extend away from Hel1, creating a C-shaped opening for substrate engagement (Fig. 1C). In the BLT dsRNA-bound state, Hel2 and Hel2i swivel toward Hel1 to clamp on the terminus (Fig. 1, D and E, and movies S1 and S2).

Within the helicase domain, density was observed for only one RNA strand, indicative of local unwinding (Fig. 1F and fig. S9C). Unwinding would likely require ATP hydrolysis and possibly was enabled by contaminating ATP in commercial preparations of ATP-γS (16). Using dsRNAs with a nick in sense or antisense strands (fig. S11A), we performed in vitro unwinding assays (fig. S11B). With ATP, dmDcr-2RIII, but not the adenosine triphosphatase (ATPase)–defective Walker A mutant dmDcr-2RIII,K34A (16), unwound BLT dsRNA termini (fig. S11B; compare lanes 3 and 7, top and bottom panels).

The unwound single strand maintained an A-form conformation (Fig. 1F), likely to minimize entropic costs of reannealing before cleavage in RNase III sites. Whether the RIG-I helicase unwinds dsRNA is controversial (26, 27), but related helicases exhibit unwinding activity (28). Local unwinding may facilitate dmDcr-2’s helicase domain in binding and translocating along dsRNA.

The Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini (2932), but our structures suggested that the helicase domain also binds termini. To investigate this idea, we purified dmDcr-2ΔHel,RIII (Fig. 2A), which lacked the helicase domain (Fig. 1A). Consistent with previous studies (16), in gel-shift assays full-length dmDcr-2RIII bound both BLT and 3′ovr dsRNA (Fig. 2B, dsRNA design; and Fig. 2C, top); ATP increased affinity for BLT dsRNA and decreased affinity for 3′ovr dsRNA (Fig. 2, C and D and table S2). However, whereas dmDcr-2ΔHel,RIII bound 3′ovr dsRNA with an affinity similar to dmDcr-2RIII, its binding to BLT dsRNA was not detected (Fig. 2C, bottom panel, Fig. 2D, and table S2). The inability of dmDcr-2ΔHel,RIII to bind BLT dsRNA was not due to the absence of ATP hydrolysis because dmDcr-2RIII,K34A bound BLT dsRNA efficiently (fig. S12, A and B, and table S2). Thus, the helicase domain is required for binding BLT, but not 3′ovr, dsRNA.

Fig. 2 dmDcr-2’s helicase domain is required to recognize and cleave BLT dsRNA.

(A) Gel-filtration and SDS–polyacrylamide gel electrophoresis analyses of dmDcr-2ΔHel,RIII. (B) Cartoon of dsRNA used in (C) to (F), showing modifications that block binding at one end. (C) Gel mobility shift assays of dmDcr-2RIII (top) and dmDcr-2ΔHel,RIII (bottom) with 52 BLT or 3′ovr dsRNA, ±5 mM ATP (n ≥ 3). (D) Binding curves using data as in (C). Data points, mean ± SD (n = 3). (E) Single-turnover cleavage assays of 52 BLT or 3′ovr dsRNA (1 nM) with dmDcr-2WT or dmDcr-2ΔHel (30 nM), ± 5 mM ATP (n = 3). Only initial cleavage is monitored, because this removes 5′ 32P. Arrow, 22-nt siRNA product. AH, alkaline hydrolysis. Left, nt lengths. (F) Quantification of cleavage assays as in (E). Percent dsRNA cleaved (all dsRNA except uncleaved) and percent siRNAs (21 to 23 nt products) resulting from first cleavage were quantified. Data points, mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Single-turnover cleavage assays showed that neither dmDcr-2WT nor dmDcr-2ΔHel (fig. S12C) cleaved BLT dsRNA without ATP (Fig. 2, E and F, lanes 5 and 6). With ATP, cleavage of BLT dsRNA by dmDcr-2WT gave heterogeneous cleavage products characteristic of Dicer enzymes with ATPase activity (16, 33), but strikingly, dmDcr-2ΔHel was incapable of cleaving BLT dsRNA (Fig. 2, E and F; compare lanes 9 and 10). As expected (15, 16), cleavage of 3′ovr dsRNA was independent of ATP and with both dmDcr-2WT and dmDcr-2ΔHel produced a single siRNA-sized 22-nucleotide (nt) band (Fig. 2E; compare lanes 7, 8, 11, and 12). dmDcr-2ΔHel cleaved 3′ovr dsRNA more efficiently than dmDcr-2WT (Fig. 2, E and F, compare lanes 7, 8, 11, and 12), suggesting that the helicase domain hinders cleavage of 3′ovr dsRNA. This observation is reminiscent of autoinhibition by hsDcr-1’s helicase domain in processing 3′ovr dsRNAs (34).

Our biochemical and structural studies indicated that dmDcr-2 has two modes of substrate recognition and cleavage: one mediated by Platform-PAZ domains and resulting in precise cleavage of 3′ovr dsRNAs into base-paired 22-nt siRNAs (22mer siRNAs) and a second mediated by the helicase domain and resulting in heterogeneous cleavage of BLT dsRNAs. We searched for amino acids that might separately affect cleavage of a 3′ovr or BLT dsRNA. We created one variant of dmDcr-2 (Fig. 1A, PP) containing five point mutations in the Platform and PAZ domains (35). Multiple crystal structures show that a phenylalanine in the C-terminal domain (CTD) of RIG-I recognizes BLT dsRNA by stacking on the terminal base pair (24). Dicer enzymes do not have a CTD, but for the second variant we searched for regions in dmDcr-2 with sequence similarity to the CTD. Within the region identified (fig. S13, A to C), we mutated a single phenylalanine to a glycine (dmDcr-2F225G).

We compared activities of purified dmDcr-2PP and dmDcr-2F225G (fig. S13D) to dmDcr-2WT using single-turnover cleavage assays (Fig. 3, A and B). As expected, cleavage of BLT dsRNA was not observed without ATP (Fig. 3, A and B, lanes 5 to 7). However, with ATP, cleavage of BLT dsRNA by dmDcr-2WT or dmDcr-2PP appeared nearly identical (Fig. 3, A and B, lanes 11 and 13), whereas cleavage was completely disrupted by the helicase point mutation in dmDcr-2F225G (Fig. 3, A and B, lane 12). [At least part of this effect is due to weakened BLT dsRNA binding (fig. S13, E and F, and table S2)]. By contrast, cleavage of 3′ovr dsRNA was independent of ATP and minimally affected by the F225G helicase mutation (Fig. 3, A and B, lanes 8, 9, 14, and 15). However, cleavage was eliminated by mutations in the Platform-PAZ domains (Fig. 3, A and B, lanes 10 and 16). These data reiterate that cleavage of 3′ovr dsRNA is mediated by Platform-PAZ domains, whereas the helicase domain coordinates recognition and cleavage of BLT dsRNA.

Fig. 3 Helicase and Platform-PAZ domains differentially contribute to cleavage of BLT and 3′ovr dsRNA.

(A) Single-turnover cleavage assays of 52 BLT or 3′ovr dsRNA (1 nM) with dmDcr-2WT, dmDcr-2F225G, and dmDcr-2PP (30 nM), ±5 mM ATP (n = 3). Substrates were as described in Fig. 2, B and E. AH, arrow, as in Fig. 2E. (B) Quantification of cleavage assays, as in (A). Data points, mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P > 0.05, n.s. (nonsignificant). (C) Substrates used in (D) and (E), with additional details in Fig. 2, B and E. In chimeras, nts 21 to 23 from the 5′ end of the sense strand, and nts 32 to 34 from the 5′ end of the antisense strand were deoxyribonucleotides (dashed circles). (D) Single-turnover cleavage assays of regular or chimeric 52 BLT or 3′ovr dsRNA (1 nM) with dmDcr-2WT (30 nM), ±5 mM ATP (n = 4). (E) Quantification of cleavage assays as in (D). Data points, mean ± SD (n = 4).

While dmDcr-2PP cleaved BLT dsRNA to yield a pattern nearly identical to dmDcr-2WT, levels of 22 nt siRNA decreased (Fig. 3, A and B, lanes 11 and 13). Because 22-nt siRNA was not observed with dmDcr-2F225G (Fig. 3A, lane 12), we hypothesized that this species derived from dsRNA that was threaded through the helicase domain until the BLT terminus encountered the Platform-PAZ domains. To confirm that smaller products (<22 nt) did not result from degradation of 22-nt siRNA, we monitored cleavage of chimeric dsRNAs containing deoxynucleotides at positions 21 to 23 from the 5′ terminus (Fig. 3C). Cleavage of 3′ovr dsRNA was eliminated with chimeric molecules (Fig. 3, D and E; compare lanes 8, 9, 15, and 16), as expected for Platform-PAZ–mediated cleavage. However, for BLT dsRNA, while 22-nt siRNA was absent, all other fragments were visible (Fig. 3, D and E; compare lanes 6 and 13), consistent with a helicase-mediated threading mechanism.

Studies of Dicer from other organisms indicate Platform-PAZ domains bind termini of 3′ovr dsRNA to allow measuring to RNase III active sites and production of an siRNA length (29, 30). Our cryo-EM structure of the dmDcr-2 complex and subsequent biochemical studies suggested that BLT dsRNA is cleaved differently, and in an ATP-dependent manner, threaded through the helicase domain to encounter the RNase III active sites. We tested the threading model by designing dsRNA with blocks at specific positions (Fig. 4A). Measurements using our EM density maps predicted that BLT dsRNAs are threaded through the helicase domain ~20 bp before encountering RNase III domains. To trap threading intermediates, we put biotin-dT analogs on both strands of 52 BLT dsRNA, at positions 28 or 37, counting from the 5′ end of the sense strand (Fig. 4A); there was no significant difference in cleavage of these modified dsRNAs (figs. S14, A and B). However, we hypothesized that the addition of streptavidin to biotin-dT–substituted dsRNAs (Block dsRNAs) would arrest threading of dsRNAs through the helicase. When dsRNA was incubated with streptavidin before initiating cleavage with dmDcr-2WT and ATP, we trapped early (<11 nt, Block-28) and intermediate (11 to 20 nt, Block-37) threading products without observing 22 nt siRNAs (Fig. 4, B and C; see fig. S14C for schematic). By contrast, cleavage by hsDcr-1 was unaffected by blocks, indicating that, at least under these conditions, hsDcr-1 cannot thread dsRNA through its helicase domain (Fig. 4D; compare lanes 12 to 14 with lanes 15 to 17).

Fig. 4 BLT dsRNA threads through helicase domain.

(A) Substrates for (B) to (D); some features described in Fig. 2, B and E. Blocked dsRNAs contained biotin-dT (red B) on both strands, with 28 and 37 indicating the position from the 5′ end of the sense strand. (B) Single-turnover cleavage assays of blocked or unblocked 52 BLT dsRNA (1 nM) with dmDcr-2WT (30 nM), 5 mM ATP, and 80 nM streptavidin (n = 3). dsRNA was preincubated with streptavidin before adding dmDcr-2WT. Arrow, AH, as in Fig. 2E. (C) Quantification of cleavage at 75 min, as in (B). dsRNA cleaved (%) is plotted based on all products (total), those >23 nt, siRNAs (21 to 23 nt products), those 11 to 20 nts, and those <11 nts. Data points, mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P > 0.05, n.s. (nonsignificant). (D) Single-turnover cleavage assays as in (B), with dmDcr-2WT or hsDcr-1WT, ±80 nM streptavidin (n = 3). Black arrow, siRNA product (22 nt) with dmDcr-2WT; green arrow, siRNA product (26 nt) with hsDcr-1WT. (E) Model for recognition and cleavage of BLT and 3′ovr dsRNA by dmDcr-2. Dotted orange arrow, clamping of helicase on BLT dsRNA; dotted white arrow, unwinding; dotted gray box, dmDcr-2RIII•BLT dsRNA•ATP-γS complex shown in Fig. 1D (see also fig. S10); red arrow, cleavage; gray arrow, threading intermediates (see text and movie S3 for details). Model for 3′ovr recognition from data reported here and elsewhere (see also movie S4) (23, 34).

We anticipated that short threading intermediates (<22 nts) might be unique to the initial cleavage event. However, threading intermediates were observed with dmDcr-2WT under multiple-turnover conditions using internally 32P-labeled dsRNAs, increasing proportionally with 22mer siRNAs through the reaction time course (fig. S14, D and E). Thus, at least in vitro, threading intermediates are recurring by-products of processive cleavage and not specific to the initial cleavage. dmDcr-2’s highly efficient, helicase-dependent, processive cleavage is likely advantageous in antiviral defense. The generation of heterogeneous cleavage products during processive cleavage is predicted to dampen the phasing signal of viral siRNAs and is consistent with the overlapping and discontinuous viral siRNAs observed in invertebrate cells (6, 13, 36).

The dsRNA binding protein (dsRBP) Loquacious-PD (Loqs-PD) allows dmDcr-2 to cleave independent of termini (16, 37) and is required for processing endogenous siRNAs (38), but not for an antiviral response (13). This suggests that dmDcr-2’s intrinsic termini preferences function in viral defense, whereas Loqs-PD allows processing of endogenous dsRNA with diverse termini. By monitoring cleavage of dsRNAs with 5′ovr termini, or overhangs on both strands (fig. S15), we determined that dsRNA with an accessible 3′ terminus is preferentially recognized by the Platform-PAZ domain and, without this feature, is processed by threading through the helicase domain.

RIG-I distinguishes capped termini of cellular transcripts from tri- and diphosphorylated termini of viral transcripts, and this is inferred to allow self versus nonself discrimination (8). We found that, like RIG-I, dmDcr-2 cannot efficiently process dsRNAs capped at the 5′ terminus, although the phosphorylation state does not affect cleavage (fig. S16, A and B, and table S3). These results may reflect dmDcr-2’s ability to process precursors of both endogenous and viral siRNAs.

We show that dmDcr-2 has two modes of cleavage (Fig. 4E and movies S3 and S4). dmDcr-2 is capable of using its Platform-PAZ domain to recognize 3′ovr dsRNAs in vitro, but it is unknown whether dmDcr-2 processes such dsRNAs in vivo. dmDcr-2’s cognate dsRBP, R2D2, may inhibit recognition and processing of substrates with 3′ovr termini (17). As such, the Platform-PAZ domains of dmDcr-2 may function solely on dsRNAs that are threaded through the helicase domain. At least in vitro, hsDcr-1 does not distinguish termini and does not exhibit helicase-dependent threading. Unlike dmDcr-2, hsDcr-1 may rely on the Platform-PAZ domain for generating viral siRNAs. Indeed, mutations to the Platform-PAZ domains of hsDcr-1 disrupt viral siRNA biogenesis (19). However, given the conservation of hsDcr-1’s helicase domain, it is intriguing to consider that, under certain conditions, perhaps with additional factors, hsDcr-1 might mediate processive cleavage by threading of dsRNA through the helicase domain.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S16

Tables S1 to S3

References (3966)

References and Notes

Acknowledgments: We thank D. Cazalla and E. Cao for critique of the manuscript and P. J. Aruscavage for technical assistance. EM was performed at University of Utah EM Core Laboratory, with computational support from Utah Center for High Performance Computing. RNA was synthesized by the DNA/Peptide facility (Health Sciences Center Cores at University of Utah). This work was supported by funding from the National Institute of General Medical Sciences (R01GM121706) and the H. A. and Edna Benning Presidential Endowed Chair (to B.L.B.). The authors declare no competing financial interests. The models and cryo-EM maps are available via the following accession numbers: Protein Data Bank (PDB) 6BUA, Electron Microscopy Data Bank (EMDB) EMD-7291, EMD-7292 (apo-dmDcr-2RIII); PDB 6BU9, EMD-7290 (dmDcr-2RIII•52 BLT dsRNA•ATP-γS complex).
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