You'd Prefer An Argonaute

Allosteric regulation of Argonaute proteins by miRNAs

Sergej Djuranovic, Michelle Kim Zinchenko, Junho K Hur, Ali Nahvi, Julie L Brunelle, Elizabeth J Rogers & Rachel Green

Nature Structural & Molecular Biology 17, 144 – 150, February 2010.
doi:10.1038/nsmb.1736

This week’s summary and analysis by David Garcia:

It’s a good thing that one of the proteins central to RNAi, Argonaute, has a beefy, determined sounding name, because it’s everywhere these days. (Fortunately the wussy sounding “P-element induced wimpy testes,” or “PIWI” proteins are only a subclass of Argonautes, and not the other way around.) Argonautes owe their celebrity status to, of course, their relationship with widespread small RNAs. Numerous groups have studied Argonaute structure and function in vitro and in vivo. This week’s paper from Rachel Green’s group I think mostly convincingly shows Drosophila Ago1 operating allosterically, through a pleasing mix of structural bioinformatics, biochemistry, and cell culture experiments.

Focusing on the MID domain of eukaryotic Argonautes (Agos), the paper begins with bioinformatic analyses demonstrating sequence and structural similarities to the ligand binding domains of bacterial proteins that exhibit allosteric behavior, namely the coupling of metabolite binding in one site to active function in another distant site. Using the CLANS program they showed that MID domains themselves clustered into groups of sequence similarity that reflect their function: inhibition of translation (via miRNAs) or mRNA cleavage (via siRNAs). Agos that they classified as involved in translational repression, including DmAgo1, HsAgo1-4, and CeAlg1-2, formed a tight cluster separate from DmAgo2 and CeRde1, both which are involved in siRNA directed repression. They saw looser clustering for other domains in Ago, like PAZ or PIWI. Thus they argue that MID domain function, reflected in sequence, largely distinguishes various Ago family members. These conclusions might be tempered by the fact that some Agos, including HsAgo2 and DmAgo1, have dual-functionality: they can mediate mRNA degradation/translational repression and have slicer activity. (A growing body of evidence is showing that mRNA degradation is a substantial part of miRNA directed repression, although this doesn’t preclude their final model.)

The authors next searched for biochemical signatures that distinguish different Agos. Running tagged, purified MID domains over m7-GTP-Sepharose resins, they observed the miRNA Agos like DmAgo1 and CeAlg-1 bound more tightly than the siRNA Agos DmAgo2 and CeRde-1. Free nucleotides could compete with binding for DmAgo2 MID, suggesting a single site in this domain that binds the 5’ nucleotide of the sRNA was responsible for binding to the resin. In contrast, for the MID of DmAgo1, free nucleotides actually stimulated binding to the cap-like structure, suggesting a second allosteric site.

They next tested full-length Agos (DmAgo2 was slightly truncated) in the presence of miRNA, and saw strongly increased affinity for the cap-like structure for DmAgo1, but not for DmAgo2, indicating some crosstalk between two distinct sites that bind the sRNA and cap-like structure. The binding affinity of DmAgo1 for the cap-like structure increased with increasing [miRNA].

Filter binding assays were used to do the inverse experiment—whether binding of a miRNA can be stimulated by addition of free nucleotides/analogs with varying resemblance to the cap-like structure. They only saw stimulated miRNA binding with tri-phosphorylated nucleotides, which most closely resembled the cap. No such effect was seen for DmAgo2. Consistent with this result, addition of miRNA duplex stimulated binding of a labeled, capped mRNA ten-fold for DmAgo1, but no-fold for DmAgo2. Free nucleotides that resembled caps could compete away the labeled mRNA.

With these pleasant in vitro results in hand, demonstrating allostery for DmAgo1 but not DmAgo2, they moved in vivo into S2 cells. But here, while supporting their hypothesis, I find the results less satisfying due to their choice of reporter system. They employed a tethered Ago reporter system (reference) that examines Ago mediated inhibition of a luciferase reporter without need for a miRNA. The utility of the system comes from the ability to directly assess the effect of mutations in the tagged and tethered Ago on the reporter, without competition from endogenous Ago. But in my opinion the compromise is too great, ignoring the effect of a targeting miRNA on Ago structure and activity.

Anyways, they saw that DmAgo1 repressed the reporter strongly while DmAgo2 did not. Then they tested mutations in conserved residues in DmAgo1 predicted to be responsible for interacting with the 5’ nucleotide of the sRNA. These mutations actually caused substantial de-repression, which as they note, was very unexpected because the Ago protein is tethered to the mRNA, so it shouldn’t matter whether or not it can bind a 5’ nucleotide. They surmise that Ago needs to bind a miRNA for full activity, even if it’s tethered. (Another possibility is that even binding free nucleotides would be sufficient, and this was still perturbed in the mutants.)

Next they pursued an exposed region in the MID domain that could bind a cap. Mutation of a specific residue caused reduced binding to m7-GTP-Sepharose resin, and complete de-repression in the reporter assay. They declared this site the second allosteric site, which binds mRNA caps. In cell lysates, they observed that only the Ago variants that repressed in the reporter assays could effectively bind miRNA, cap structures, and GW182, a protein that has been implicated in miRNA directed repression.

So overall I found the bioinformatics and in vitro data very convincing, but for the in vivo data, despite the fact that it supports the allosteric model, I’m less enthusiastic because of the experimental system.

A burning question arising from their allosteric model is how the cap binds Ago. I wouldn’t naturally assume that the cap would be free and close to Agos moving along 3’ UTRs. Messenger RNAs are most efficiently translated when they’re circularized by protein-protein-RNA interactions between the PABPs and proteins that bind the cap. Could Ago or some other co-factor then destabilize these interactions to compete for the cap? Would this change be a prerequisite for Ago mediated repression? How reversible would the exchange be? What about mRNAs with multiple target sites–each message has only a single cap but may host multiple Agos? As the authors imply in the discussion section, Ago binding the cap may only be favorable when RISC is bound to an authentic target (potentially avoiding indiscriminate activation of Ago by the caps of other mRNAs floating nearby that aren’t necessarily themselves actively repressed), so there may be a kinetic argument to be made. Finally, has the allostery of DmAgo1, and potentially other Agos, co-evolved with its preference for miRNAs that usually target the 3’ UTR, a region perhaps physically closer to the cap of a circularized mRNA? Hopefully Rachel Green’s group and others are asking these questions. We crave answers.