Volume 3, Issue 6, Pages 1893-1900 (June 2013) Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity  Kotaro Nakanishi, Manuel Ascano, Tasos Gogakos, Satoko Ishibe-Murakami, Artem A. Serganov, Daniel Briskin, Pavel Morozov, Thomas Tuschl, Dinshaw J. Patel  Cell Reports  Volume 3, Issue 6, Pages 1893-1900 (June 2013) DOI: 10.1016/j.celrep.2013.06.010 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

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 depicted in a ribbon representation. Guide RNA positions 1–8, 20, and 21 (red) are traceable in the structure of the complex. (B) The 5′-end nucleotide recognition is depicted. Residues involved in RNA recognition are depicted 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 (A). Only the first nucleotide is shown for clarity. (C) A kink is introduced in the bound guide RNA strand by the insertion of I363 on helix 7. (D) Trajectories of hAGO1- and hAGO2-bound guide RNAs. The guide RNAs bound to hAGO1 and hAGO2 are colored in red and blue, respectively. (E) Solvent-exposed seed nucleotides 2–4 in the binary complex of guide-RNA-bound hAGO1, the latter depicted with a surface representation. The color codes are the same as those in (A). Helix 7 and I363 are shown as transparent representations. (F and G) Guide RNA recognition on the 2′ OH groups (F) and backbone phosphate oxygens (G). The guide RNA is depicted as a ball-stick representation. Residues involved in interactions with the guide RNA are depicted as stick representations. The 2′ OH groups are depicted as white-colored spheres in (F), whereas water molecules are depicted as blue-colored spheres. See also Figures S1 and S2. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Structural Comparison between hAGO1 and hAGO2 (A) Structural resemblance between hAGO1 and hAGO2 upon 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 with 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, which contains a pair of bound tryptophans (left panel). Different amino acid 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 and E) Different local structures of the cS cluster. cS1 (orange), cS3 (red), cS7 (blue), and cS10 (green) form a cluster at the edge of the nucleic-acid-binding channel of hAGO1 (D) and hAGO2 (E). The N-terminal amino acids 36–64 are colored in magenta. The directionality of cS7 for hAGO1 (D, top view) and hAGO2 (E, top view) are highlighted with arrows. The different local structures composed of cS1, cS3 and cS10 for hAGO1 (D, bottom view) and hAGO2 (E, bottom view) are highlighted with black circles on their surface representations. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

Figure 3 cS7 Loop of hAGO1 Serves to Sterically Hinder Guide-Target Accommodation in the Nucleic-Acid-Binding Channel (A) hAGO1 modeled with the 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 and 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 (B) and hAGO2 (C). The surface representations are depicted as transparent. The black arrowhead points to the potential clash between hAGO1-cS7 and the bound guide RNA strand in (B). The color code for cS insertion elements is the same as in (A). (D and E) Proposed model for RNA target cleavage for hAGO1 (D) and hAGO2 (E). In hAGO1, the cS7 element protrudes toward the guide-target duplex, thereby having an impact on cleavage efficacy after the release of the target strand from the PAZ domain during the propagation step (D). In hAGO2, the cS7 element is recessed and does not have an impact on target cleavage. The DEDR and DEDH catalytic tetrads are shown as hexagons. For clarity, only the PAZ, MID, and PIWI domains are depicted in (D) and (E). Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

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 indicated with a dotted circle. Important residues on cS7 and the catalytic tetrad are depicted with 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-nucleotide-long RNA, complementary to the let-7a guide, was used as a cleavage substrate. The cleavage site is located across from the 10th and 11th nucleotide from the 5′ end of the guide RNA, yielding a 9-nucleotide-long product. Guide RNA load-normalized quantitation, by fraction cleaved, is shown. H, alkaline hydrolysis ladder of 5′-labeled target RNA. See also Figure S4 and Table S3. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

Figure S1 RNA Electron Density and Ramachandran Plot Analyses, Related to Figure 1 (A) Simulated-annealing Fo-Fc omit map contoured at 3.5σ around the bound guide RNA. hAGO1 is drawn as a ribbon model. (B and C) Ramachandran plot analyses of (B) the current structure of hAGO1 at 2.3 Åresolution and (C) the previously determined crystal structure of hAGO2 at 2.3 Å resolution. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Nuclease Treatment of Isolated Nucleic Acids from Recombinant hAGO1 and hAGO2 Proteins, Related to Figure 1 Nucleic acids co-purified from hAGO1 and hAGO2 proteins were isolated, dephosphorylated, radiolabelled, and then subjected to DNase I or RNase A treatment. A 22-nt long radiolabelled synthetic deoxyribonucleotide was also used for comparison. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Sequence Alignment of Human AGOs, Related to Figures 2 and 3 The four catalytic tetrad residues are highlighted with yellow background. α helices and β strands are depicted by sticks and arrows, respectively. cS regions are underscored. The color code of the cS1, cS3, cS7, and cS10 is the same as in Figures 2D, 2E, and 3A–3C. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions

Figure S4 Purification of hAGO Proteins, Related to Figure 4 AGO proteins isolated from baculoviral-infected High Five insect cells were fractionated by SDS-PAGE and stained with colloidal blue coomassie, demonstrating their comparable purity. Cell Reports 2013 3, 1893-1900DOI: (10.1016/j.celrep.2013.06.010) Copyright © 2013 The Authors Terms and Conditions