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. 2015 Oct 30;43(19):9418-33.
doi: 10.1093/nar/gkv937. Epub 2015 Sep 17.

Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly

June Hyun Park  1 , Chanseok Shin  2
Affiliations

Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly

June Hyun Park et al. Nucleic Acids Res. .

Abstract

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila.

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Figures

Figure 1.
Figure 1.
Small RNA maturation is highly sensitive to the ambient temperature both in vitro and in vivo. (A) Small RNA duplexes used in this study; miR-1 with an endogenous duplex structure, functionally asymmetric miR-1 siRNA (miR-1 was perfectly paired to its antisense, except for the first position from the 5′ end) and miR-1 siRNA PS (miR-1 siRNA introduced with phosphorothioate linkage at the scissile phosphate, denoted by a star). The guide and passenger-strands are shown in black and red, respectively. (B) PS modification inhibits the cleavage of the passenger-strand. Small RNA duplexes containing radiolabeled passenger-strands were incubated in lysates expressing tagged AGO2 for the indicated times at 25°C. (C) Distinct efficacy of target cleavage at different temperatures for each different small RNA duplex. Small RNAs duplexes were assembled in lysates expressing tagged AGO2 for 15 min at the indicated temperature (mock refers to the no duplex control). Cap-radiolabeled target RNA was then added and further incubated for 15 min at the indicated temperatures. (D) Distinct efficacy of unwinding at different temperatures for each different small RNA duplex. Small RNA duplexes carrying radiolabeled guide strands were incubated in lysates from expressing tagged AGO2 for 30 min at the indicated temperature. Small RNAs incorporated into the AGO2 complexes were immunopurified and analyzed in native gel, along with the 10% input (IN) relative to the IP. (E) Small RNA maturation is dependent on the ambient temperature in living cells. HEK293T cells were co-transfected with 10 nM of small RNA duplexes and FLAG-AGO2 plasmids. Cells were then cultured either at 28°C or 37°C and harvested at 48 h post-transfection; cell lysates were subjected to FLAG-IP, followed by northern blotting with the miR-1 probe. The blot was probed for U6 snRNA as a loading control. Ethidium bromide-stained 5S rRNA served as another loading control. The numbers below the blot are the relative expression levels, normalized using the U6 snRNA loading control. (F) Cell lysates prepared as in panel (E) were used for the target cleavage assay. Cap-radiolabeled target RNA was added and incubated for the indicated times.
Figure 2.
Figure 2.
Slicer-dependency is determined by temperature and the Mg2+ level. (A) The effect of temperature and Mg2+ on the degree of passenger-strand cleavage. siRNA duplexes containing radiolabeled passenger-strands were incubated in lysates expressing tagged AGO2 for 15 min either at 25°C or 37°C, with Mg2+ concentrations ranging from 0 to 5 mM. The incubation with 5 mM EDTA served as a negative control. (B) A schematic of the analysis for (C–F). (C–F) The slicer-dependency of RISC assembly decreases with increasing temperature and decreasing Mg2+ levels. Small RNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged AGO2 at the indicated temperature and Mg2+ concentrations for the indicated times. Under the same condition, target cleavages were analyzed after 30 min of small RNA assembly. Representative data of at least two independent experiments are shown.
Figure 3.
Figure 3.
Slicer-deficient AGO proteins can unwind the siRNA duplex in a temperature-dependent manner. (A–D) PIWI catalytic mutant AGO2 is capable of unwinding the siRNA duplex. Small RNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged AGO2 (D597A) at the indicated temperature and Mg2+ concentrations for the indicated times. Representative data of at least two independent experiments are shown. (E) Higher temperature drives the unwinding of the siRNA duplex when slicer-assisted pathway is not available. Left: Small RNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged AGO2 (D597A) at the indicated temperature and Mg2+ concentrations for the indicated times. Right panel: 30 min of RISC assembly at the indicated temperature using a temperature gradient in a PCR machine. (F–H) Non-slicer AGO protein can unwind the siRNA duplex at the physiological temperature of humans. Small RNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged AGO1 at the indicated temperature and Mg2+ concentrations for the indicated times. Representative data of at least two independent experiments are shown. (I) The siRNA duplex is only functionally active at 37°C. Deadenylation by AGO1-RISC was monitored in lysates expressing tagged AGO1 at the indicated temperature and 3.75 mM Mg2+ at the indicated times.
Figure 4.
Figure 4.
Temperature per se is not sufficient to induce duplex unwinding, but requires prior AGO loading. (A) A schematic of the experimental design. ssRNA, but not dsRNA, can be incorporated efficiently into purified human AGO2 protein in the absence of chaperone proteins. If the duplex is spontaneously unwound at 37°C, the resulting ssRNA might then be loaded into the purified AGO2 protein. (B) None of the duplexes spontaneously unwound at 37°C and form a complex with the purified AGO2 protein. Radiolabeled ssRNA or duplexes (with radiolabeled guide strands) were incubated with increasing concentrations of recombinant hAGO2 for 30 min at the indicated temperatures. Recombinant hAGO2 forms active complexes only with ssRNA. Representative data of at least two independent experiments are shown. (C) Prior duplex association with the AGO protein is necessary for duplex unwinding. siRNA duplexes carrying radiolabeled guide strands were incubated with lysates expressing tagged AGO2 for 1 h at 15°C, where duplex loading, but not unwinding, is permissible. Pre-RISCs were subsequently immunopurified at 4°C and washed six times with either low salt (150 mM) or high salt (1 M) before shifting the temperature to 37°C.
Figure 5.
Figure 5.
Effects of duplex thermodynamic stability on slicer-independent unwinding. (A) 2′-OMe modified small RNA duplexes used in this study; miR-1-OMe and miR-1 siRNA-OMe (full 2′-OMe-modified passenger-strand) and miR-1 siRNA-OMe9 (with 2′-OMe modification only at the ninth of the passenger-strand). Unmodified and 2′OMe-modified nts (denoted by Nm) are shown in black and red, respectively. (B) 2′-OMe-modification completely blocks the cleavage of the passenger-strand. Small RNA duplexes containing radiolabeled passenger-strands were incubated in lysates expressing tagged AGO2 for the indicated times at 25°C in the presence of 5 mM Mg2+ to ensure efficient passenger-strand cleavage. Unmodified siRNA served as a positive control. (C and D) The degree of RISC maturation at different temperatures depends on duplex stability. Small RNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged AGO2 at the indicated temperature and Mg2+ concentrations for the indicated times. Under the same conditions, target cleavages were analyzed after 30 min of small RNA assembly. Representative data of at least two independent experiments are shown. (E) Slicer-independent unwinding is strictly temperature-dependent. Radiolabeled slicer-resistant duplex (siRNA–OMe9) was first incubated at 15°C for 1 h to assemble the pre-RISCs. Subsequently, a 20-fold excess of non-radiolabeled siRNA-OMe9 duplex (cold-competitor) was added to prevent further incorporation of the radiolabeled duplex (at 0 min) before shifting the temperature to 37°C. (F) RISC maturation is closely correlated with parameters that affect duplex stability. RISC assembly was monitored for the highly stable duplex (siRNA–OMe) in lysates expressing tagged AGO2 at the indicated temperature and Mg2+ concentrations at the indicated times.
Figure 6.
Figure 6.
Slicer-independent unwinding is mediated by the functional domains of the AGO protein. (A) Left panel: A structural representation of the AGO protein during target RNA recognition. The guide strand (red) binds to its target (light blue) from the 5′ seed-portion (nucleation) and extends to form a double helix (propagation) that terminates at position 16, which is blocked by the N domain. During the propagation step, the 3′ end of the guide is released from its anchor site in the PAZ domain with the help of pivotal movement of AGO. Right panel: Slicer-independent unwinding is assumed to be the reverse process of target RNA recognition, with the passenger-strand being a target. The N and PAZ domains might play a role in duplex unwinding because the N domain blocks additional pairing (beyond 16th) at the 3′ end, which could be further disrupted by dynamic 3′ anchoring of the PAZ domain. (B and C) N mutant (F181A) is defective for both slicer-dependent and -independent unwinding. Small RNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged wild-type and N mutant (F181A) AGO2 proteins at the indicated temperature and Mg2+ concentrations for the indicated times. A higher temperature could partially compensate for the wedging defect. Representative data of at least two independent experiments are shown. (D) PAZ truncation causes a severe defect in slicer-independent unwinding. miRNA duplexes containing radiolabeled guide strands were incubated in lysates expressing tagged wild-type or PAZ-truncated AGO2 proteins at the indicated temperature and Mg2+ for the indicated times. Note that the size of the ΔPAZ-RISC complexes shifted downward due to the truncation of the PAZ domain. (E) Quantitation of (D). Data are mean ± SD for two independent experiments. (F) Functional domains of AGO2 are required for unwinding, but AGO2-mediated slicer-activity per se is dispensable at the physiological temperature of humans. Slicer-resistant duplex (siRNA–OMe9) containing radiolabeled guide strand was incubated in lysates expressing tagged wild-type, PAZ truncation (ΔPAZ), N mutant (F181A) and PIWI catalytic mutant (D597A) AGO2 proteins at the indicated temperature and Mg2+ concentration for the indicated times.
Figure 7.
Figure 7.
Slicer-independent unwinding is a conserved mechanism. (A–D) Distinct slicer-dependency at different temperatures in Drosophila AGO2. Small RNAs were assembled in S2 cell lysates only for 5 min (to avoid early saturation) at the indicated temperature and Mg2+ level. Cap-radiolabeled target RNA was then added and incubated for the indicated times at the indicated temperatures and Mg2+ concentrations. Quantitation of the fraction target cleaved (%) is shown below the gel image. Black: siRNA with 3.75 mM Mg2+, Brown: siRNA-OMe9 with 3.75 mM Mg2+, Gray: siRNA with 0.75 mM Mg2+, Red: siRNA-OMe9 with 0.75 mM Mg2+. Data are mean ± SD for at least two independent experiments. (E) A propose model for small RNA maturation in human RISCs. miRNAs are unwound most efficiently in all four human AGOs through the aid of internal mismatches. Slicer-deficient AGOs (1, 3 and 4) are able to unwind the highly complementary siRNA in a temperature-dependent manner. AGO2 can additionally utilize its slicer-activity for the highly complementary siRNA with slicer-dependency being positively correlated to the Mg2+ concentration and negatively correlated to temperature.
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