Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg 2+ -facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide–target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3′-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures. How Argonaute slices mRNA One of the means by which RNA silencing downregulates gene expression is through small RNA-directed cleavage of a complementary mRNA. Argonaute family proteins are essential regulators of gene expression in plants and animals. They act by binding to single-stranded guide RNA molecules to form RISC complexes that catalyse splitting of the mRNA just where the sequence is complementary to the Argonaute-bound RNA. The crystal structure of several Thermus thermophilus Argonaute proteins mutated to inhibit cleavage activity have now been determined, in complex with a guide RNA and target RNAs of different lengths. Comparison of the various structures reveals conformational changes as the guide and target RNAs pair and suggests a mechanism for cleavage involving three aspartates and two Mg 2+ ions. The Argonaute (Ago) family of proteins provides the slicer activity of the RNA-induced silencing complex, with the Ago component of the complex providing the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here, the crystal structures of ternary complexes of Thermus thermophilus Ago catalytic mutants are reported and analysed.