Figure 1. Core Features of miRNA and siRNA Silencing.
(A) Common aspects of all miRNA and siRNA pathways. Double-stranded RNA precursors of various kinds are processed by a Dicer protein into short (∼20−30 nt) fragments. One strand of the processed duplex is loaded into an Argonaute protein, enabling target RNA recognition through Watson-Crick base pairing. Once the target is recognized, its expression is modulated by one of several distinct mechanisms, depending on the biological context.
(B) Dicer proteins cleave dsRNA precursors into characteristic lengths through the action of two RNase III domains. The domain arrangement of most Dicer enzymes is shown at the top. Processing occurs most readily at dsRNA ends, which associate with the PAZ domain present in most Dicer enzymes. The substrate is then positioned within the active sites of the RNase III domains, which cleave the ∼20−30 nt miRNA/siRNA duplex from its precursor. This model is supported by the crystal structure of Giardia Dicer, shown with a dsRNA modeled into the structure (image kindly provided by J. Doudna). In addition to the canonical PAZ and RNase III domains, the structure shows active-site metal ions (purple) and a “ruler” helix (red) that helps to specify the length of the siRNA product.
(C) Argonaute proteins are RNA silencing effectors that are guided to their targets by short single-stranded nucleic acids. The canonical arrangement of Ago domains is given at the top. Below is a crystal structure of the Thermus thermophilus Ago protein, with a bound DNA guide strand base paired to an RNA target. The 5′ end of the guide strand associates with a binding pocket in the Mid domain, and the 3′ end binds the PAZ domain. The target cleavage site is juxtaposed with active-site residues in the PIWI domain, though in this case cleavage is suppressed by mismatches between the guide and the target. (Structure reprinted with permission from Macmillan Publishers Ltd.: Wang et al. [2008]. Nature 456, 921−926.)