Structural Foundations of RNA Silencing by Argonaute

Abstract

Nearly every cell in the human body contains a set of programmable gene-silencing proteins named Argonaute. Argonaute proteins mediate gene regulation by small RNAs and thereby contribute to cellular homeostasis during diverse physiological process, such as stem cell maintenance, fertilization, and heart development. Over the last decade, remarkable progress has been made toward understanding Argonaute proteins, small RNAs, and their roles in eukaryotic biology. Here, we review current understanding of Argonaute proteins from a structural prospective and discuss unanswered questions surrounding this fascinating class of enzymes.

Keywords: Argonaute; RNA silencing; RNA-induced silencing complex; microRNA.

Figure 1. Architecture of Argonaute

A. Linear schematic of the Argonaute primary sequence. Numbers correspond to residue numbers in human Ago2. Regions that contact the 5′ and 3′ ends of the small guide RNA are indicated. B. Surface representation of human Ago2 bound to an siRNA guide (red). C. Close up view of the binding site for the guide RNA 5′ end. D. Close up view of the seed region prior to target binding shows the majority of the seed splayed out in preparation for pairing to target RNAs. E. Surface representation of Ago2 reveals that only guide nucleotides g2–g4 are fully available for initiating interactions with target RNAs. F. Close up view of the binding site for the guide RNA 3′ end in the PAZ domain.

Figure 2. Model for Argonaute targeting

A. Argonaute searches for target sites, in part via lateral diffusion, along single stranded regions of potential target RNAs. B. Semi-stable interactions are made between target and nucleotides g2–g4 of small RNA guide. C. Stable interactions are made when target (t) nucleotides match the full guide RNA seed (g2–g8). Adenosine nucleotides in the t1 position further stabilize the ternary complex through interactions with a surface pocket in Ago2. Full seed pairing requires movement of a-helix-7, which shifts to dock into the minor grove of the guide-taget duplex (lower panel). D. The shift in helix-7 also opens the central cleft, exposing guide nucleotides in the supplemental region (g13–g16) for interactions with target RNAs.

Figure 3. Structural features of human Ago2

A. Close up view of the slicer active site in Ago2 reveals a magnesium ion (yellow-orange sphere) coordinated to Ago2 and 4 water molecules (blue spheres). Comparison with T. thermophilus (purple) indicates the magnesium ion is in an inactive position prior to target binding (right panel). B. Tryptophan-binding pockets in the Ago2 PIWI domain. C. Positions of post-translations modifications mapped onto the Ago2 crystal structure. Phosphoration sites are indicated in green. Prolyl hydroxylation site indicted in blue.

Figure 4. Comparison of Argonaute and Piwi structures

A. Surface representation of human Ago2 (PDB 4W5N, right) alongside silkworm Piwi (PDB 5GUH, left). Both proteins share a common domain architecture. However, differences in the positions of the N and PAZ domains results in a substantially wider and more open central cleft in Piwi. B. The Piwi seed region is less constrained and ordered prior to target binding than the Argonaute seed region.