LA JOLLA, Calif.—With their sights set on learning tocontrol natural gene-silencing processes, a team of scientists led by TheScripps Research Institute (TSRI) recently published a study showing how toboost or inhibit Argonaute 2, a gene-silencing mechanism that normally servesas a major controller of cells activities.
The U.S. National Institutes of Health-funded study, "HighlyComplementary Target RNAs Promote Release of Guide RNAs from Human Argonaute2," was published in the May 9 issue of the journal Molecular Cell. According to the TSRI researchers, who conductedthe study along with the NovartisInstitutes for Biomedical Research, their findings could pave the way for thedevelopment of an entirely new approach to treating human disease, oneinvolving a powerful new class of drugs designed to treat viral infections andeven cancer.
Ian J. MacRae, assistant professor in TSRI's Department ofIntegrative Structural and
Computational Biology and a principal investigator for thestudy, explains that Argonaute 2 is a "molecular machine" in cells that cangrab and destroy the RNA transcripts of specific genes, preventing them frombeing translated into proteins. Argonaute 2 and other Argonaute proteinsregulate the influence of about a third of the genes found in humans and othermammals, and thus are among the most important modulators of our cells'day-to-day activities. Argonaute's gene-silencing functions also help cellscope with rogue genetic activity from invading viruses or cancer-promoting DNAmutations.
"Argonaute2 is a catalytic engine of mammalian RNAi," saysMacRae. "It is loaded with small RNA or siRNA, and we can use small RNA as aguide to locate and destroy, or downregulate, any miRNA that has acomplementary sequence. In a sense, it is a programmable nuclease, a mechanismof post-transcriptional regulation."
However, Argonaute's complex workings are not yetunderstood. Before Argonaute starts a "search-and-destroy" mission against aspecific type of target RNA, an Argonaute 2 protein takes on board atarget-recognition device—a short length of guide RNA, or microRNA (miRNA). ThemiRNA's sequence is mostly complementary to the target RNA, so that it canstick tightly to it. How exactly the Argonaute protein and its miRNA guide thenmanage to part company, however, has eluded researchers who have encountereddifficulty separating Argonaute proteins from miRNA in a lab dish, says MacRae.
"A lot of work has been done to date that has determined thestructure of these things. It's clear from biology that some small microRNA arestable, but some are less stable than others. It also turns out that in thepast decade, people have found that some siRNA are more stable than others andlast longer. But the basis for understanding that differential stability was amystery," MacRae says.
In an initial set of experiments, the scientistsdemonstrated that when an miRNA hooks up with an Argonaute 2, the pair do remainlocked together and functioning for an exceptionally long time—days to weeks,whereas solo miRNA normally is degraded within minutes. They confirmed thatdecoy RNA designed to match miRNA this way can hasten the miRNA's unloadingfrom Argonaute, effectively dialing down these miRNA's normal gene-silencingactivities. By contrast, mismatches at one end delayed unloading, actuallyenhancing the gene-silencing activity.
"We started by seeing how stable the complex is, and itturns out, it's very stable in vitro,"MacRae explains. "We then looked for conditions that would destabilize it. Whenwe put in complementary RNA, it destabilizes the complex significantly. Then wewent on and characterized what the requirements were, and did our best to seeif it could happen in living cells, which are very different than a chemicallab.
"I think from a mechanistic standpoint, what we're reallyseeing is loading in reverse," he continues. "I think this can be exploited intwo ways. The hope is that if we can better understand this relationship, youcan begin to control the lifetime of small RNA living in cells. You can makethem long-lasting, or not long-lasting.
But MacRae admits, "the next thing we're trying to figureout is how all that works. We have some guesses, but no clear answer."
MacRae's lab, which published a study that year describingthe use of X-ray crystallography to determine the first high-resolutionstructure of an Argonaute2-miRNA complex, is now working on a structural studyof 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 unloadingprocess," said MacRae. "I also hope that the basic research we're doing cancontribute 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 aspecific tissue is harder. I expect that the first successes here will be intissues that are amenable to delivery—liver cancer, hepatitis, etc. Then, asdelivery methods become more sophisticated, it will be possible to target othertissues as well," he says.