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


RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes1,2. Argonaute proteins3, 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.

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Figure 1: General features of AfPiwi–siRNA interactions and distortion of the 5′ terminus of the RNA duplex.
Figure 2: Interactions between AfPiwi and the guide RNA strand.
Figure 3: Proposed model for a 19-nucleotide guide–target duplex bound to AfPiwi.

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We are grateful to staff at ESRF for help with data collection.The work was funded by Cancer Research–UK, the Wellcome Trust and the Institute of Cancer Research.

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Correspondence to David Barford.

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Supplementary information

Supplementary Figure S1

Shows the conformation of the siRNA duplex in the two copies of the AfPiwi-RNA complex per P1 unit cell (a), and a superimposition of the siRNA duplex onto an A-form RNA helix (b). (PPT 363 kb)

Supplementary Figure S2

Shows a multiple sequence alignment of the PIWI domains of AfPiwi, PfAgo and selected eukaryotic Argonautes. (PPT 1405 kb)

Supplementary Figure S3

Shows a superimposition of AfPiwi with the target RNA strand and PfAgo in the region of the putative slicer catalytic site. (PPT 639 kb)

Supplementary Table S1

Table of crystallographic data collection, processing and refinement statistics. (DOC 22 kb)

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Parker, J., Roe, S. & Barford, D. Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex. Nature 434, 663–666 (2005).

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