Abstract
Argonaute is the principal protein component of the mechanisms of RNA silencing, providing anchor sites for the small guide RNA strand and the 'slicer' activity for cleavage of target mRNAs or short passenger RNA strands. Argonaute is the core constituent of the silencing effector complexes RISC (RNA-induced silencing complex) and the RITS complex (RNA-induced initiation of transcriptional gene silencing complex), interacting directly or indirectly with Dicer proteins, R2D2/Loquacious/TRBP and GW182 family proteins in the former, and Chp1 and Tas3 in the latter. In a breakthrough series of papers, Patel et al. provide a set of 'molecular snapshots' of the catalytic cycle of Argonaute, exploiting mismatches and mutants to capture and visualize by X-ray crystallography Argonaute from Thermus thermophilus with guide and target strands at various stages of the silencing process. The structural studies, coupled to structure-directed biochemical analysis, together with other thermodynamic and kinetic studies, provide insights into Argonaute with implications for the mechanisms of RNA silencing in eukaryotes.
Highlights
Genetic and biochemical studies first implicated Argonaute as a key component of the mechanisms of RNA silencing in eukaryotes [1]
Argonaute proteins fall mainly into two subfamilies (Ago and Piwi), defined initially on the basis of sequence similarity [1], with an additional subfamily specific to Caenorhabditis elegans and outliers, which include the prokaryotic Argonautes, though the latter category displays some similarity to the Piwi subfamily
The structural studies show that three-dimensional Argonaute is roughly a bi-lobal protein, with an N-terminal lobe composed of an N-domain, L1 linker region and PAZ domain and a C-terminal lobe composed of MID and PIWI domains
Summary
Genetic and biochemical studies first implicated Argonaute (or Ago) as a key component of the mechanisms of RNA silencing in eukaryotes [1]. It is quite striking that there are almost no direct hydrogen-bonding contacts from TtAgo to the target strand across the whole length of the substrate (apart from around the scissile phosphate, which are likely to function to fine-tune positioning of the target with respect to the catalytic residues in the Ago scaffold.) Second, as already discussed, both ends of the formed guide/target duplex are splayed by Ago, which provides a starting point for unwinding. One of these free single-stranded ends may be the initial substrate for an ATP-assisted helicase to mediate unwinding. The extraordinary rearrangements in the Ago scaffold coupled to duplex propagation, evidenced by the pivoting of the Ago domains around an uninterrupted, undistorted guide/target duplex, suggest tensions accumulated and overcome during propagation which may be exploited to eject the sliced target strand, once the duplex is compromised by the central slicing event
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