Insights into the mechanism of RNA interference have now been provided by three papers, two in Nature and one in Nature Structural & Molecular Biology. Small interfering RNAs (siRNAs) — 19–23-base-pair duplexes with 5′-phosphorylated ends and 3′ overhangs — are produced by the enzyme Dicer. They enter the RNA-induced silencing complex (RISC), where a single-stranded guide RNA from the duplex functions to select target mRNAs for silencing. RISC comprises the RNA guide and an Argonaute protein, and the latter contains an N-terminal PAZ domain and a C-terminal PIWI domain. Recent studies on the PAZ domain have indicated that it is involved in recognizing the 3′ overhang, but what is the role of the PIWI domain?
In the first paper, Barford and colleagues describe the 2.2-Å-resolution crystal structure of a complex between a Piwi protein from Archaeoglobus fulgidus (AfPiwi) and an siRNA-like duplex. This duplex mimics the 5′ end of a guide RNA bound to a 3′-overhanging mRNA target. They found that AfPiwi contains a highly conserved metal-binding site that binds to the phosphorylated 5′ end of the 'guide' RNA and distorts it, such that it is separated from the complementary nucleotide on the 'target' strand. The complementary nucleotide and the 3′ overhang exit the protein through a short channel. The remaining nucleotides adopt an A-form helix that extends along a channel in AfPiwi. AfPiwi makes extensive, non-sequence-specific interactions with the guide RNA, but only a few contacts with the target RNA, which is consistent with the need for target dissociation following mRNA cleavage. Using modelling to extend the A-form helix, the authors found that the scissile bond in the target RNA is positioned adjacent to the putative catalytic slicer site.
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