The Making of a Slicer: Activation of Human Argonaute-1 Christopher R. Faehnle, Elad Elkayam, Astrid D. Haase, Gregory J. Hannon, Leemor Joshua-Tor  Cell Reports  Volume 3, Issue 6, Pages 1901-1909 (June 2013) DOI: 10.1016/j.celrep.2013.05.033 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Structure of hAgo1 in Complex with let-7 (A) The overall structure of hAgo1 in complex with let-7 guide RNA. The individual domains of hAgo1 are labeled and color coded. let-7 miRNA is shown as an orange cartoon. Nucleotides 1–10 stretch from the Mid domain and pass through L2, the PIWI domain, and L1. A dashed line indicates the projected path of the disordered nucleotides 11–20. Nucleotides 21 and 22 are modeled in the PAZ domain. (B) A magnified view of the path of the seed region of let-7 bound to hAgo1. The let-7 miRNA bound to hAgo1 is shown as orange sticks. The superimposed miR20a from the hAgo2-miR20a complex (PDB 4F3T) is shown as gray sticks. Structural elements are colored according to the domain colors in (A). Residues that make kinks in the let-7 miRNA are shown as sticks. See also Figure S1. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Effect of Argonaute Domains on Slicer Activity (A) Structural superposition of hAgo1 and hAgo2 on the basis of the Mid-PIWI lobe. hAgo1 is shown as a cartoon with the same color scheme as Figure 1A. hAgo2 is shown as a gray cartoon. (B) The same alignment as shown in (A) but with a 90° rotation around the horizontal axis. All of the domains are colored as in (A), but the Mid and PAZ domains are deleted for clarity. Secondary structure elements in the N domain that interact with the L1, L2, and PIWI domains are indicated as are the elements composing the subdomain of the N that move in hAgo1 with respect to hAgo2. (C) Slicer assays for WT hAgo1, hAgo2, the mutants of the catalytic tetrad, and chimeric hAgo2 constructs swapped with inactive hAgo1 domains. (D) Slicer assays of hAgo1 chimeras comprised of single and double domain swaps from hAgo2. (E) A plot of fraction product cleaved over a 60 min time course showing enhanced slicing by the hAgo2 N domain. The standard error deviation from the mean of three individual replicates is plotted with data points connected by a smooth curve. (F) Slicer assays for hAgo3-hAgo2 N domain chimera. Time points were taken every 10, 30, and 60 min. A line designates an irrelevant lane that was cropped. Slicer assays shown in (C), (D), and (F) are representative of at least three individual replicates. See also Figure S2. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Two Mutations in the PIWI Domain Activate hAgo1 (A) A view of the superimposed active sites in the PIWI domain of hAgo1 and hAgo2. The PIWI domain of hAgo1 is colored purple, and hAgo2 is colored gray. Conserved active site residues are labeled with yellow text. Mutations in hAgo1 that activate slicing are labeled in green. The PIWI domain loops (PL1, PL2, and PL3) that cluster near the active site are indicated. (B) Sequence alignment focused on the catalytic tetrad residues for hAgo1, hAgo2, hAgo3, Drosophila Ago1 and Ago2 (dAgo1 and dAgo2), and two other eukaryotic Argonautes for which structures are known, NcQDE-2 and KpAgo. Conserved catalytic residues are highlighted in yellow, and mutations that activate hAgo1 are highlighted in green. The PIWI domain loops PL2 and PL3 are indicated. (C) Slicer assays showing activated hAgo1H by the mutation of L674F. The N domain of hAgo2 enhances the slicing by the activated hAgo1. F676L mutants of hAgo2 have a severe defect in slicing. (D) A plot of fraction product cleaved over a 60 min time course showing the activated hAgo1H L674F and the enhancement from the hAgo2 N domain. The standard error deviation from the mean of three individual replicates is plotted with data points connected by a smooth curve. (E) Mutants of PL3 loop residues in hAgo2 show that E673 is important for slicing. (F) Mutants of F676 in hAgo2 greatly impact slicing activity. Slicer assays shown in (C), (E), and (F) are representative of at least three individual replicates. See also Figure S3. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure 4 The PL3 Loop Plays a Role in Slicing (A) Structure of hAgo1 with a modeled A-form duplex RNA. Domains of hAgo1 are colored as in Figure 1A. The PAZ domain and L1 linker are omitted for clarity. let-7 miRNA is shown in orange, the modeled guide RNA is shown in red, and the target strand is shown in blue. The location of the scissile phosphate is indicated with scissors. The active site tetrad is shown as yellow sticks. The PL3 loop is highlighted green, and important residues identified in this study are shown as green sticks. (B) A close-up view of (A) with only the PIWI domain shown. The PL2 and PL3 loops are indicated. (C) A-form RNA modeled in hAgo2 with the same layout as (B). See also Figure S4. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Interactions between hAgo1 and let-7 miRNA and endoRNA, Related to Figure 1 (A) Summary of interactions between hAgo1 and the bases, phosphate backbone and 2′-OH of the let-7 miRNA. Amino acid residues are colored according to the domain color scheme from Figure 1A. (B) Hydrogen bonds (red dash) and ion pair (blue dashes) interactions between hAgo1 and the 5′-U of let-7. (C) 5′-end binding pocket in the hAgo1-endoRNA complex. FO-FC difference map prior to inclusion of RNA contoured at 2.5 σ is shown in blue. 5′-A1 modeled in the syn conformation is shown as orange sticks. All of the RNA interactions in the 5′-binding pocket are as indicated in (B) except a new interaction is observed between the side-chain of Y813 and the adenine base. (D) Small RNAs bound to hAgo1-3 were 5′-32P radiolabeled and separated by denaturing 15% PAGE. (E) Size profile of small RNAs bound to purified hAgo1-3 or present in SF9 cells expressing hAgo1 (input) were analyzed by Illumina sequencing. The range of two biological replicates is indicated. (F) First nucleotide preference of 20nt small RNAs bound to hAgo1-3 or present in input extracts. The average of two biological replicates (p values < 0.05) is depicted. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Overall Structure of hAgo1 with endoRNA and let-7 miRNA along with Domain Comparison with hAgo2, Related to Figure 2 (A) Structure of hAgo1-let-7 complex with the domains colored as in Figure 1A. The FO-FC difference map prior to inclusion of RNA in the model contoured at 2.5 σ is shown in blue. Nucleotides 1-10 and 21-22 are modeled as sticks. (B) Structure of hAgo1-endoRNA complex with an identical layout as in (A). Nucleotides 1-9 and 19-20 are shown as sticks. (C–H) Superposition of each isolated domain of hAgo1 onto hAgo2. Domains are labeled and colored as shown in Figure 1A. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Guide RNA Binding Assay and SDS-PAGE Analysis of Argonaute Mutants, Related to Figure 3 (A) The indicated Argonaute construct was loaded with 5′-32P radio-labeled miR20 guide RNA using the same loading conditions as described in the Experimental Procedures. To assay for guide RNA loading efficiency the proteins were immobilized on nitrocellulose membranes with a slot-blot apparatus (Schleicher and Schuell). Guide RNA alone failed to bind the nitrocellulose membrane, but was retained in all of the Argonaute mutants described in this study. (B) SDS-PAGE analysis of purified Argonaute mutants. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions

Figure S4 Sequence Alignment of hAgo1, hAgo2, and hAgo3, Related to Figure 4 Secondary structure elements extracted from the hAgo1 structure are shown above the sequences and colored according to the domains of hAgo1 as shown in Figure 1A. Conserved amino acids are in black and nonconserved amino acids are shaded blue. The location of the PL1, PL2 and PL3 loops are labeled. Residues that make up the catalytic tetrad are indicated with a red star below. The two point mutations made in hAgo1 that activate slicing are shaded green. Cell Reports 2013 3, 1901-1909DOI: (10.1016/j.celrep.2013.05.033) Copyright © 2013 The Authors Terms and Conditions