Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex

Abstract

RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes. Argonaute proteins, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5' end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5' nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5' nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3' overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute-siRNA guide complex.

Figure 1

General features of AfPiwi-siRNA interactions and distortion of the 5′ terminus of the RNA duplex. (a) Schematic of the RNA duplex co-crystallized with AfPiwi, indicating nomenclature for guide and target strands. (b) Orthogonal views of the AfPiwi-RNA complex. The divalent metal in the G1 binding pocket is shown as a red sphere (M). (c) Two orthogonal views of the RNA 2Fo-Fc electron density map contoured at 1α. Left: view of the RNA as seen by the protein. G1 to G9 (G1 to G11 right) and T(-2) to T9 are placed in electron density. Figure generated using PYMOL (http://www.pymol.com).

Figure 2

Interactions between AfPiwi and the guide RNA strand. (a) Detailed view of the G1 binding pocket. Side chains coloured grey show conformations of selected residues in the absence of RNA. The concerted shift of Tyr 123 and Lys 127 suggests an induced fit mechanism for phosphate binding. The divalent metal (M) is shown as a yellow sphere. (b) View of interactions with the phosphate groups of G1 to G5. The target strand is omitted for clarity.

Figure 3

Proposed model for a 19 nucleotide guide - target duplex bound to AfPiwi. (a) View showing the positioning of the scissile phosphate of the target strand (between T10 and T11, coloured red) with respect to the location of the putative slicer catalytic site. The modelled RNA region is coloured darker for both strands (G10 to G19 and T10 to T19). PIWI sub-domains are shown. Catalytic Asp residues of the DDE motif, are absent in AfPiwi. (b) Stereoview with the molecular surface of AfPiwi colour-coded according to structural conservation (Supplementary Fig. 2). CRI, CRII and CRIII (Conserved Regions I, II and III) are indicated.