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. 2013 Jun 27;3(6):1893-900.
doi: 10.1016/j.celrep.2013.06.010.

Eukaryote-specific insertion elements control human ARGONAUTE slicer activity

Kotaro Nakanishi  1 , Manuel Ascano, Tasos Gogakos, Satoko Ishibe-Murakami, Artem A Serganov, Daniel Briskin, Pavel Morozov, Thomas Tuschl, Dinshaw J Patel
Affiliations

Eukaryote-specific insertion elements control human ARGONAUTE slicer activity

Kotaro Nakanishi et al. Cell Rep. .

Abstract

We have solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify changes that evolutionarily rendered hAGO1 inactive, we compared our structure with guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop, forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad with R805H led to low-level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions P670S and P675Q substantially augmented hAGO1 activity. Evolutionary amino acid changes to hAGO1 were readily reversible, suggesting that loading of guide RNA and pairing of seed-based miRNA and target RNA constrain its sequence drift.

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Figures

Figure 1
Figure 1
Crystal Structure of hAGO1. (A) Structure of hAGO1 bound to guide RNA. hAGO1 with N (cyan), L1 (yellow), PAZ (magenta), L2 (gray), MID (orange), and PIWI (green) domains is drawn in a ribbon representation. Guide RNA positions 1–8 and 20–21 (red) are traceable in the structure of the complex. (B) 5′-end nucleotide recognition. Residues involving the RNA recognition are shown in a stick representation. A water molecule (blue) is shown as a sphere. The O2′ and O4′ sugar atoms are colored in white and cyan, respectively. Hydrogen bonds are shown as dotted lines. The other color codes are the same as in panel A. Only the first nucleotide is shown for clarity. (C) A kink is introduced in the bound guide RNA strand by insertion of I363 on helix 7. (D) Trajectories of hAGO1- and hAGO2-bound guide RNAs. The guide RNAs bound to hAGO1 and to hAGO2 are colored in red and blue, respectively. (E) Solvent-exposed seed nucleotides 2–4 in the binary complex of guide-RNA-bound hAGO1, with the latter shown in a surface representation. The color codes are as in panel A. Helix 7 and I363 are shown as transparent representations. (F, G) Guide RNA recognition on the 2′ OH groups (panel F) and backbone phosphate oxygens (panel G). The guide RNA is shown as a ball-stick representation. Residues involved in interactions with the guide RNA are depicted in stick representations. The 2′-OH groups are shown as white-colored spheres in panel F, while water molecules are drawn as blue-colored spheres. See also Figures S1 and S2.
Figure 2
Figure 2
Structural Comparison between hAGO1 and hAGO2. (A) Structural resemblance between hAGO1 and hAGO2 following superposition of their structures. The current hAGO1 (blue) and the tryptophan-bound hAGO2 (yellow) (PDB ID: 4EI3) structures are superposed on their MID-PIWI lobes. The two tryptophans (red) observed in tryptophan-bound hAGO2 are depicted in stick representations. (B) The plausible tryptophan-binding pockets on the PIWI domain of hAGO1. The local structures around the tryptophan-binding pockets are quite similar between hAGO2 and hAGO1, as shown for hAGO2 containing a pair of bound tryptophans (left panel). Different amino acids residues between hAGO1 and hAGO2 are colored in yellow on the surface representation of hAGO1 (blue) (right panel). (C) Structural difference within cS7 between hAGO1 and hAGO2. The cS7 element is shown in a dashed ellipse with hAGO1 in blue and hAGO2 in yellow. (D, E) Different local structures of the cS cluster. The cS1 (orange), cS3 (red), cS7 (blue), and cS10 (green) form a cluster at the edge of the nucleic-acid-binding channel of hAGO1 (panel D) and hAGO2 (panel E). The N-terminal amino acids 36–64 are colored in magenta. The directionality of cS7 for hAGO1 (panel D, top view) and hAGO2 (panel E, top view) are highlighted with arrows. The different local structures composed of the cS1, cS3 and cS10 for hAGO1 (panel D, bottom view) and hAGO2 (panel E, bottom view) are highlighted with black circles on their surface representations.
Figure 3
Figure 3
cS7 Loop of hAGO1 Serves to Sterically Hinder Guide-Target Accommodation in the Nucleic-Acid-Binding Channel. (A) hAGO1 modeled with guide-target RNA duplex. The model results from superposition of the current guide RNA-hAGO1 binary complex and the TtAgo guide-target ternary complex as superpositioned on their MID-PIWI domains. hAGO1 (white) is shown as a ribbon representation. The modeled guide and target are colored in red and slate, respectively. (B, C) The cS7 insertion element of hAGO1 potentially clashes with the bound guide RNA strand. The expanded boxed segments highlight the surface around the cS7 of hAGO1 (panel B) and hAGO2 (panel C). The surface representations are shown as transparent. The black arrowhead points to the potential clash between hAGO1-cS7 and the bound guide RNA strand in panel B. The color code for cS insertion elements is the same as in panel A. (D, E) Proposed model for RNA target cleavage for hAGO1 (panel D) and hAGO2 (panel E). In hAGO1, the cS7 element protrudes towards guide-target duplex, thereby impacting on cleavage efficacy following release of the target strand from the PAZ domain during the propagation step (panel D). In hAGO2, the cS7 element is recessed, and does not impact on target cleavage. The DEDR and DEDH catalytic tetrads are shown as hexagons. Only the PAZ, MID and PIWI domains are depicted in panels D and E for clarity.
Figure 4
Figure 4
Cleavage Activity of hAGO1 Mutants. (A) Alignment of four catalytic residues in hAGO1, hAGO2, hAGO3 and hAGO4. (B) Structural difference in insertion element cS7 between hAGO1 (blue) and hAGO2 (yellow) drawn in ribbon representation. The cS7 of hAGO1 is highlighted in dotted circle. Important residues on cS7 and the catalytic tetrad are depicted in stick representation. (C) Cleavage activity of hAGO1 mutants. Baculovirus-expressed and purified recombinant hAGO proteins were loaded with 5′ phosphorylated guide RNAs representing mature hsa-let-7a sequence. Radiolabeled 21-nt RNA, complementary to the let-7a guide was used as cleavage substrate. The cleavage site is located across of the 10th and 11th nucleotide from the 5′ end of the guide RNA, yielding a 9-nt product. Guide-RNA load-normalized quantitation, by fraction cleaved, is shown. Abbreviation: H, alkaline hydrolysis ladder of 5′ labeled target RNA. See also Figure S4 and Table S3.
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