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TSRI’s Argonaute 2 finding unravels some mysteries about gene silencing
LA JOLLA, Calif.—With their sights set on learning to control natural gene-silencing processes, a team of scientists led by The Scripps Research Institute (TSRI) recently published a study showing how to boost or inhibit Argonaute 2, a gene-silencing mechanism that normally serves as a major controller of cells activities.
The U.S. National Institutes of Health-funded study, "Highly Complementary Target RNAs Promote Release of Guide RNAs from Human Argonaute 2," was published in the May 9 issue of the journal Molecular Cell. According to the TSRI researchers, who conducted the study along with the Novartis Institutes for Biomedical Research, their findings could pave the way for the development of an entirely new approach to treating human disease, one involving a powerful new class of drugs designed to treat viral infections and even cancer.
Ian J. MacRae, assistant professor in TSRI's Department of Integrative Structural and
Computational Biology and a principal investigator for the study, explains that Argonaute 2 is a "molecular machine" in cells that can grab and destroy the RNA transcripts of specific genes, preventing them from being translated into proteins. Argonaute 2 and other Argonaute proteins regulate the influence of about a third of the genes found in humans and other mammals, and thus are among the most important modulators of our cells' day-to-day activities. Argonaute's gene-silencing functions also help cells cope with rogue genetic activity from invading viruses or cancer-promoting DNA mutations.
"Argonaute2 is a catalytic engine of mammalian RNAi," says MacRae. "It is loaded with small RNA or siRNA, and we can use small RNA as a guide to locate and destroy, or downregulate, any miRNA that has a complementary sequence. In a sense, it is a programmable nuclease, a mechanism of post- transcriptional regulation."
However, Argonaute's complex workings are not yet understood. Before Argonaute starts a "search-and-destroy" mission against a specific type of target RNA, an Argonaute 2 protein takes on board a target- recognition device—a short length of guide RNA, or microRNA (miRNA). The miRNA's sequence is mostly complementary to the target RNA, so that it can stick tightly to it. How exactly the Argonaute protein and its miRNA guide then manage to part company, however, has eluded researchers who have encountered difficulty separating Argonaute proteins from miRNA in a lab dish, says MacRae.
"A lot of work has been done to date that has determined the structure of these things. It's clear from biology that some small microRNA are stable, but some are less stable than others. It also turns out that in the past decade, people have found that some siRNA are more stable than others and last longer. But the basis for understanding that differential stability was a mystery," MacRae says.
In an initial set of experiments, the scientists demonstrated that when an miRNA hooks up with an Argonaute 2, the pair do remain locked together and functioning for an exceptionally long time—days to weeks, whereas solo miRNA normally is degraded within minutes. They confirmed that decoy RNA designed to match miRNA this way can hasten the miRNA's unloading from Argonaute, effectively dialing down these miRNA's normal gene- silencing activities. By contrast, mismatches at one end delayed unloading, actually enhancing the gene-silencing activity.
"We started by seeing how stable the complex is, and it turns out, it's very stable in vitro," MacRae explains. "We then looked for conditions that would destabilize it. When we put in complementary RNA, it destabilizes the complex significantly. Then we went on and characterized what the requirements were, and did our best to see if it could happen in living cells, which are very different than a chemical lab.
"I think from a mechanistic standpoint, what we're really seeing is loading in reverse," he continues. "I think this can be exploited in two ways. The hope is that if we can better understand this relationship, you can begin to control the lifetime of small RNA living in cells. You can make them long-lasting, or not long-lasting.
But MacRae admits, "the next thing we're trying to figure out is how all that works. We have some guesses, but no clear answer."
MacRae's lab, which published a study that year describing the use of X-ray crystallography to determine the first high-resolution structure of an Argonaute2-miRNA complex, is now working on a structural study of the complex as it grabs a target RNA.
"When we can see the structural details of that interaction, then I think we¹ll have a much better handle on this loading and unloading process," said MacRae. "I also hope that the basic research we're doing can contribute to the development of small RNA-based therapeutics."
One challenge to overcome is delivery, concludes MacRae.
"If you are working with cells in vitro, it works really well. Delivering them to target a specific tissue is harder. I expect that the first successes here will be in tissues that are amenable to delivery—liver cancer, hepatitis, etc. Then, as delivery methods become more sophisticated, it will be possible to target other tissues as well," he says.