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MIT, MSKCC Team Reports Structure of Yeast Argonaute, Shedding Light on RNAi Process

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A team of researchers from the Massachusetts Institute of Technology and Memorial Sloan-Kettering Cancer Center this month reported on the crystal structure of a eukaryotic Argonaute protein, offering new insights into the molecules that drive the RNA interference process.

"You can learn a lot from biochemical experiments, but to more fully understand a protein like Argonaute, it's useful to know where all of the atoms are and which amino acids are playing important roles," MIT researcher and study co-author David Bartel said in a statement. "Learning the Argonaute crystal structure is an important step in understanding the RNAi biochemical pathway and will be the basis for many future experiments."

During the RNAi process, the RNase III enzyme Dicer cuts double-stranded RNA into siRNAs, which are then incorporated into Argonaute. The passenger strand is cleaved and discarded, leaving the RNA-induced silence complex, which uses the guide strand to drive interactions with target RNAs. Argonaute then helps cleave these target RNAs, completing the RNAi process.

Despite discoveries related to the nucleation, propagation, and cleavage steps of the Argonaute, or AGO, catalytic cycle in prokaryotes such as the bacteria Thermus thermophilus, “the physiological role of
prokaryotic AGOs is enigmatic,” with the origin of the guide DNA unknown, and the "absence of recognizable RNAi pathway components in bacteria,” Bartel and his colleagues wrote in the June 21 issue of Nature.

“Therefore, attention has turned to eukaryotic AGOs, which use RNA guides and have protein-binding partners absent in bacteria,” and are also larger than their prokaryotic counterparts because of insertion elements of unknown structure and function, they added.

Still, the structural characterization of the entire AGO protein has “remained a challenge.”

To address this issue, the investigators turned to the budding yeast >Kluyveromyces polysporus, in which they had previously determined the structure and mechanism of Dicer.

In the Nature paper, the investigators reported on the structure of K. Polysporus AGO “fortuitously complexed with guide RNA originating from small RNA duplexes autonomously loaded and processed by recombinant” K. Polysporus AGO.

“Despite their diverse sequences, guide-RNA nucleotides 1 [to] 8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 29-hydroxyl groups pre-organizing the backbone of nucleotides 2 [to] 8 in a near-A-form conformation,” they wrote.

“Compared with prokaryotic Argonautes, [K. Polysporus AGO] has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide–target pairing.

“Compared with Argonautes in inactive conformations, [K. Polysporus AGO] has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket,” they added. “Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.”

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