Structural basis for microRNA targeting

Abstract

MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. We determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing. Pairing to nt 2 to 5 promotes conformational changes that expose nt 2 to 8 and 13 to 16 for further target recognition. Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, whereas an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. These results explain the conserved nucleotide-pairing patterns in animal miRNA target sites first observed over two decades ago.

Fig. 1

Structure of the Ago2-guide complex. (A) Schematic of the Ago2 primary sequence. Front and top views of human Ago2 bound to a defined guide RNA (red). Ago2 contains a large central cleft between two lobes (N-PAZ and MID-PIWI) connected by two linker domains (L1 and L2). (B) Guide RNA omit map contoured at 2 σ (blue mesh). (C) Nucleotides g2-g5 are fully exposed, while Ago2 occludes nucleotides g6 and g7. (D) The 3′ half of the guide is treaded through the N-PAZ channel. (E) View down the N-PAZ channel.

Fig. 2

Structure of Ago2 bound to seed-matched target RNAs. (A) Sequences of guide (red) and target RNAs (blue). (B) Front and top views of Ago2 bound to guide and target RNAs. (C) Binding pocket for t1 adenine between L2 and MID domains. (D) Equilibrium binding data for target RNAs bearing different t1 nucleotides. Mean values from ≥ three independent replicates ± standard error shown. (E) Ago2 interrogates the guide-target minor groove. Protein is shown as a ribbon, RNA in surface representation, and interacting side-chains as sticks with dots. Helix-7 (α7) indicated.

Fig. 3

Structural analysis of seed-pairing. (A-C) Ago2-guide-target complexes with pairing to g2–g7 (A), g2–g8 (B), or g2–g9 (C). (D) Alignment of g2–g9 structure (guide, red; target, blue) with g2–g7 structure (guide pink, target light blue). (E and F) Dissociation constants of wild type (WT) and F811A Ago2 proteins binding target RNAs with various degrees of guide complementarity. Ago2 was loaded guide RNAs derived from either Sod1 (E) or miR122 (F). Mean of independent triplicates, ±SEM.

Fig. 4

Ago2 restricts guide-target pairing beyond the seed. (A) Phe-811 stacks against t9 of short target RNAs. (B and C) The central cleft viewed from the MID (B) and N domain (C), showing narrowing of the cleft after g8 (g13–g16 omitted for clarity). (D–E) Ago2-guide complex bound to target RNAs paired to g2–g8 that end at t9 (D) or t11 (E). (F) Superposition of structures in (D) and (E).

Fig. 5

Comparison of Ago2-guide and Ago2-guide-target structures. (A) Helix-7 (α7) shifts to accommodate pairing to target RNAs. The Ago2-guide structure (gray) aligned to the Ago2-guide-target structure (colored). (B) The PAZ domain and helix-7 move as a rigid body. Superposition of protein components from Ago2-guide (semi-transparent) and Ago2-guide-target (opaque) structures. Arrows indicate movement from guide-only to guide-target structures. Dashed line marks hinge in the L1/L2 domains. (C-D) Contacts to the guide RNA supplemental region in the guide-only (C) and target-bound (D) structures. (E) The supplemental region (g13-g16) adopts an exposed helical conformation in the Ago2-guide-target structure. (F) Cartoon model for seed plus supplemental pairing.

Fig. 6

Inactive magnesium ion in the Ago2 active site. (A and B). Magnesium ion (green) is bound to the Asp-597 carboxylate side chain, the Val-598 main chain carbonyl, and four water molecules (brown spheres). 2F_o-F_c map (blue mesh) contoured at 1.5 σ (A), and F_o-F_c magnesium omit map (green mesh) at 15 σ (B). (C) Active site of Ago2 (gray) aligned with unplugged active site of TtAgo (yellow) (PDB ID 3DLH). (D) Ago2 active site aligned with plugged-in TtAgo (blue) (PDB ID 3HVR). Metals ions shown as spheres. (E) Ago2 active site aligned with B. halodurans RNase H (pink with red magnesium ion; PDB ID 2G8H). (F) Alignment of Ago2 (gray with green magnesium), TtAgo (blue) and B. halodurans RNase H (pink) shows the Ago2 magnesium shifted 1.5 Å from the active position.