Human Argonaute3 has slicer activity

Abstract

Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3 and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes that bind target mRNAs and silence gene expression through translational repression and deadenylation but not cleavage. Here, we report that recombinant AGO3 loaded with miR-20a cleaves complementary target RNAs, whereas AGO3 loaded with let-7a, miR-19b or miR-16 does not, indicating that AGO3 has slicer activity but that this activity depends on the guide RNA. Our cleavage assays using chimeric guides revealed the significance of seed sequence for AGO3 activity, which depends specifically on the sequence of the post-seed. Unlike AGO2, target cleavage by AGO3 requires both 5'- and 3'-flanking regions. Our 3.28 Å crystal structure shows that AGO3 forms a complete active site mirroring that of AGO2, but not a well-defined nucleic acid-binding channel. These results demonstrating that AGO3 also has slicer activity but with more intricate substrate requirements, explain the observation that AGO3 has retained the necessary catalytic residues throughout its evolution. In addition, our structure inspires the idea that the substrate-binding channel of AGO3 and consequently its cellular function, may be modulated by accessory proteins.

Figure 1.

In vitro cleavage assays of AGO2 and AGO3. (A) SDS-PAGE analysis of the recombinant AGO2 and AGO3 purified from insect cells and used in the functional studies depicted in panels D and E. The gel was stained with Coomassie blue. (B) Native mass spectra of recombinant AGO2 and AGO3. Molecular weights with standard deviations for the main species as well as the charge states are indicated. The molecular weights correspond to those expected for the AGO proteins with bound RNA. (C) Guide and target RNAs used in D–F. The cap-labels are depicted as black spheres. (D) RNA Cleavage by AGO2 and AGO3 with different guide RNAs. Top: representative gel images of in vitro cleavage assay with the recombinant AGO2 and AGO3. The cap-labeled targets were incubated with either of 1 μM recombinant AGO2 or AGO3 for 1 h and resolved on 16% (w/v) polyacrylamide/7 M urea PAGE. Bottom: average of three experiments are shown in bar graphs with error bars calculated as standard deviation. (E) A representative gel image of in vitro cleavage assay with the recombinant AGO2 (1–5 μM) and AGO3 (1–25 μM). The cap-labeled 60-nt target of miR-20a was incubated with either of recombinant AGO2 or AGO3 for 1 h and resolved on 16% (w/v) polyacrylamide/7M urea PAGE. (F) A representative gel image of in vitro cleavage assay with the catalytic mutant of immunopurified FLAG-tagged AGO2 and AGO3 as well as their wild-type counterpart. Lysates of HEK293T cells expressing either of FLAG-tagged proteins were pre-incubated with a siRNA-like duplex of miR-20a for 2 h, followed by immunoprecipitation with anti-FLAG beads. The purified RISCs were incubated with the cap-labeled 60-nt target of miR-20a for 60 min. The reactions were resolved on a 16% (w/v) polyacrylamide/7 M urea PAGE.

Figure 2.

Influence of the seed and post-seed regions on the slicer activity of AGO3. (A) In vitro slicer activities of recombinant AGO2 and AGO3 with miR-20a and let-7a. Aliquots of the reaction were sampled at 0, 10, 20, 40 or 60 min. Their slicer activities with let-7a and miR-20a are drawn as blue and red curves, respectively. (B) RNA cleavage of AGO2 (left) and AGO3 (right) loaded with chimeric guides. Either of the seed 2–4, 5–8 or 2–8 of miR-20a was incorporated into let-7a. Nucleotides of let-7a and miR-20a are colored in blue and red, respectively. (C) RNA cleavage of AGO2 (left) and AGO3 (right) loaded with chimeric guides. Either of the central or 3′-supplementary site of miR-20a was incorporated into let-7a. The color codes are the same as B. (D) RNA cleavage of AGO2 (left) and AGO3 (right) loaded with miR-17 families. The post-seed regions are shown in different colors. The different nucleotides are colored in black. (E) RNA cleavage by AGO2 with miR-19b and by AGO3 with chimeric guide seed 2–8. The mean ± standard deviation for three biological replicates is shown. Student's t-test values are shown in B–E.

Figure 3.

AGO3 has an immature nucleic acid binding channel. (A and B) Right: overall structure of AGO3-RISC (A, current structure: PDB ID: 5VM9) and AGO2-RISC (B, PDB ID: 4OLA). The bound guide RNA is depicted as a ribbon model (red). The catalytic residues are drawn as stick models (green). Left: cross-section (black) of the surface model along the nucleic acid-binding channel (white). The bound guide RNAs are colored in red. Structural differences due to the disordered motifs and the widths of their channel exit are highlighted with orange circles and black lines, respectively. The loose (AGO3) and tight (AGO2) connections between the N and L1/L2 domains are highlighted with orange arrowheads. (C and D) Structural differences around the N domain between AGO3 (C) and AGO2 (D). Disordered regions in the motif II are shown as dotted lines (orange). Residues unique to AGO3 N domain are shown as sticks (dark blue).

Figure 4.

Difference in the structural rigidity between AGO3 and AGO2. (A and B) Packing of nonpolar residues between the motifs I and II in AGO3 (A) and AGO2 (B). Van der Waals surface areas are depicted as dots. For clarity, the bound guide RNA and part of the L2 domain are not shown. (C–E) Surface models of the AGO3 NCS molecules 1 (C) and 2 (D), and AGO2-RISC (PDB ID: 4OLA) (E). The color codes are the same as Figure 3. For clarity, bound RNAs are not shown.

Figure 5.

AGO3 requires flanking regions to cleave the target sufficiently. (A) Cap-labeled (black sphere) targets of miR-20a used in B and C. Two targets, Δ5′ and Δ3′, were designed by deletion of the 5′ upstream and 3′ downstream flanking sequences from the 60-nt target of miR-20a (intact). (B and C) Influence of flanking regions on the target cleavage by AGO2 (B) and AGO3 (C). The mean ± standard deviation for three biological replicates is shown. Student's t-test values are shown in B and C.