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Scripps Team Publishes Data on Structures Behind microRNA Targeting

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NEW YORK (GenomeWeb) — A team of scientists from the Scripps Research Institute this week reported the crystal structure of a microRNA bound to human Argonaute 2 both before and after their engagement with a target mRNA, showing how the protein starts off by exposing only a small portion of its guide RNA as the complex searches for targets.

The research also identified a key aspect of how an miRNA/Ago2 complex uses a metal ion to prevent degradation of a target in the absence of extensive complementarity in favor of repressing its translation.

Since miRNAs were first discovered decades ago, significant advances have been made in understanding the structural basis for how they work. In 2006, for example, the lab of Jennifer Doudna at the University of California, Berkeley, published the crystal structure of Dicer, showing how the enzyme acts as a "molecular ruler" that recognizes dsRNA and cleaves it at specific locations.

Leading that work was Ian MacRae, now an associate professor at Scripps, who later reported on how small RNAs disrupt RISC in order to accelerate the release of guide strands from Ago2, as well as the crystal structure of human Ago2.

His latest effort, which was published in Science, took things a step further, revealing the structure of Ago2 bound to a guide RNA — in this case, an miRNA — both with and without target RNAs representing miRNA recognition sites.

Using a technique developed by Phillip Zamore and colleagues at the University of Massachusetts Medical School that allows for the isolation of Ago2 linked with a specific guide RNA, MacRae and his team were able to get a clear look at how a miRNA sits within Ago2 before and after they engage a target.

The scientists found that prior to finding a target mRNA, Ago2 exposes only four nucleotides — 2 through 5 — of its guide RNA to identify candidate target sites. Doing so, they wrote in Science, "limits spurious interactions with cellular RNAs by confining pairing potential" to just a few nucleotides at first. Once that occurs, Ago2 undergoes conformational changes that expose nucleotides 2 through 8, then 13 through 16, to allow for further target recognition.

"Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner," they stated, "whereas an adenosine binding-pocket opposite guide [nucleotide] 1 further facilitates target recognition."

To MacRae, the process resembles the strategy one used to solve a word seek puzzle. "You don't look for 'elephant' at once, [for example], you look for 'ele' or 'pha'," he said. "It's a simple string search algorithm … [and] Argonaute works in the same way."

While miRNAs typically act to repress the translation of their targets, when perfect complementarity exists between them, Ago2 takes on the endonucleolytic slicing role observed in the RNAi process. This, the Scripps team noted, suggests that Ago2 has at least one other conformational state to accommodate guide-target pairing beyond an miRNA's seed region.

The mechanisms underlying slicing specificity in humans is not fully elucidated, but an observation made by MacRae and his colleagues suggest that it is regulated by an inhibitory coordination of one catalytic magnesium ion within Ago2.

Based on their experiments, it appears that slicing requires the ion be in a specific position over the target RNA. When there is only partial complementarity between a target and a guide RNA, the ion is out of its catalytic position, preventing cleavage. When there is extensive pairing between a guide RNA and its target, as occurs with siRNAs, the ion is shifted into an activated position and enables slicing, MacRae said.

Beyond contributing to the field's overall understanding of miRNAs and how they function, MacRae noted that the work may also provide insights into how better to design drugs that target the non-coding RNAs.

A high-resolution view of how Ago2 interacts with miRNAs and their targets can enable drug developers to take a more rational approach to the design of miRNA inhibitors, which have largely been developed through empirical studies, he said.

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