Argonaute Gymnastics May Guide siRNA Design (Journal Club)

Argonautes are at the heart of all RNAi-related small RNA-regulated processes. A structural understanding of how they load, activate, and utilize siRNAs to seek out and repress target mRNAs would therefore prove invaluable for the rational design of potentially ever more potent and specific siRNAs. The high-impact journal Nature appears to agree, as it has been publishing at an astonishing frequency collaborative work from the Patel and Tuschl labs at Rockefeller on beautiful structures of Argonautes at the various stages of their catalytic cycle.

In the latest publication in this series, they catch Argonaute with the activated guide strand bound to first a short (12 nucleotides), and then an elongated target RNA (15 nucleotides and longer). What is remarkable is that the enzyme undergoes a substantial structural rearrangement when binding to the longer target, most notably accompanied by the popping out of the guide strand 3’ end from the PAZ pocket where it usually sits in the single-stranded form. This is thought to reflect a 2-state mode of binding fully complementary target RNAs and may differ in terms of structural re-arrangement to what happens in the setting of microRNA target recognition which is the cause for siRNA off-targeting. What is always neat to see in those structures is how they rationalize a lot of preceding molecular biology and bioinformatics work. In this case, this includes the importance of the ‘seed’ nucleotides 2-8, the sensitivity of RNAi target cleavage to mismatches and modifications, and the relative unimportance of the 3’ terminal guide bases 17-21.

The structure may help to expand on work that shows that it is possible to design small RNAs for which target cleavage and translational repression can be functionally separated. Previous work by Dharmacon (now part of Thermo Fischer) for example showed that methylation at position 2 of the guide strand abrogates its ability to translationally repress targets, while leaving cleavage intact. While those siRNAs should be highly specific, the somewhat slow adoption of this strategy indicates to me that more work needs to be done so that similar strategies are more generalizable. Based on the two-state model and present structure, such modification-induced distortions may critically affect seed-binding energy on which translational repression is so much dependent, while the conformational shift that accompanies the recognition of fully complementary on-targets, may lock the Argonaute-guide-target complex into a stable cleavage-competent conformation. The structure may help define universally applicable modification strategies that are targeted towards the 5’ end of the guide strand.

The work also demonstrates how target cleavage is sensitive to modifications around the cleavage site, and finds certain positions better tolerate them than others. This may prove to be important for aiding siRNA activation which is thought to primarily function via cleavage of the passenger strand. Finally, the series of Argonaute structures with bound guide strand provided some nice snapshots of how the 5’ end is nestled in the so-called MID-domain of the enzyme. It is not far-fetched to believe that by optimizing this interaction (e.g. 5’ end modification, nature of the base itself etc), the capture and longevity of the guide strand in the Argonaute protein can be increased with possibly increased duration of activity of the activated enzyme complex, and in the case of single-stranded RNAi, improved loading, too. Finally, this type of structural work is also highly relevant for research into how to reconcile the need for modifications for increasing siRNA stability and avoiding innate immune responses with RNAi activity.

The next major structural insight that should guide design strategies for therapeutic RNAi will be from the elucidation of mammalian Argonaute structures (humans have 4 Argonautes), as the present work focused on archaebacterial Argonautes due to the relative ease of working with them. These, however, will differ in several important aspects from mammalian Argonautes, for example in that they utilize DNA as guides and maybe even as their natural targets. Nevertheless, with such healthy progress on the molecular understanding of RNAi, the efficiency of the RNAi Therapeutics drug development platform will only increase, and it is fun to imagine what it could look like in 10 years, at which point we may be able to watch a movie on the life of an Argonaute protein by the Patel lab.