Discovering the Building Blocks of RNA Interference
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Profiles provide insights into the lives, backgrounds, career paths, and futures of scientists who serve as Experts on ACS Chemical Biology’s online Ask the Expert feature. Readers are encouraged to submit questions to the Experts at www.acschemicalbiology.org. The editors will post the most interesting exchanges on the website.
Published online April 21, 2006 10.1021/cb600152p CCC: $33.50 © 2006 by American Chemical Society
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f all the tools biologists and chemists have in their kits, few have revolutionized these disciplines as much as RNA interference (RNAi). Also known as RNA silencing or gene silencing, this technique knocks down the function of targeted genes when double-stranded RNA called small interfering RNA (siRNA) enters cells. RNAi has been on the scene for just over a mere decade, but it has already transformed the power that researchers wield over genes. Rather than taking the costly and lengthy route of engineering knockout animals to understand a gene’s function, investigators can now quickly and cheaply accomplish that feat by using RNAi to decrease any given gene’s mRNA. Researchers are also gradually harnessing the benefits of dialing down the influence of genes involved in diseases. Even with its widespread use, scientists know little about the natural processes in cells that guide gene silencing. Phillip Zamore, a biochemist and developmental biologist at the University of Massachusetts Medical School in Worcester, MA, is intensively detailing RNAi’s major players and determining how they orchestrate gene expression. These efforts are gradually elucidating the mechanistic aspects of RNAi. Putting the Pieces Together Zamore was born in 1963 in East Flatbush, a neighborhood in Brooklyn, NY. His father, who was a lawyer, and his mother, who is a speech pathologist, encouraged an early and enduring love for reading and writing. Another of Zamore’s childhood passions was for the Lego building toy, an interest that still remains with him today. “My approach to biology is still the same approach I took to Lego as
a child—how the pieces go together and why they do or don’t work when they go together in different ways,” he says. “It’s a very reductionist approach.” In grade school, Zamore indulged this exploratory attitude by spending nearly all his free time asking myriad questions of his school’s science teacher. Later, in junior high school, he regularly exercised his curiosity in the natural world through science classes and after-school clubs. When he was 16, Zamore attended a summer program in Roswell Park, NY, geared toward nurturing young scientists. There, he did experiments to examine how glycosylation affects cultured human cells’ response to interferon. It was the first time that Zamore had ever lived on his own away from home, and he reveled in his newfound freedom. “I associated being grown up with doing science,” he says. By the time he began his undergraduate studies at Harvard University (Cambridge, MA), a school he picked for its liberal arts offerings in addition to its storied reputation, Zamore says that he was certain that he wanted to be a scientist. Though Zamore’s parents were supportive, they were often puzzled by his scientific bent. “Where I came from, nobody placed a high premium on science. It was important in life to read the classics of American literature, but knowing what a ribosome is was not something that was valued,” he says. “My father struggled to read Scientific American to figure out why I was so interested.” Seeking to maintain a well-rounded background, Zamore completed his undergraduate studies in 1985 with a Bachelor of Arts degree. He then took a job as a technician in molecular biologist Michael Green’s lab w w w. a c s c h e m i ca l biology.org
while his girlfriend, now his wife, completed her undergraduate degree. Green encouraged Zamore to pursue graduate studies in his lab at Harvard, so for the next six years, Zamore investigated the function of U2AF, a protein integral for RNA splicing (1 ). He completed his doctoral degree in 1992. Later that year, Zamore accepted a position at developmental geneticist Ruth Lehmann’s lab at Massachusetts Institute of Technology’s (MIT) Whitehead Institute in Cambridge, MA. Zamore worked with Lehmann for the next two years to investigate the molecular mechanisms behind how pole plasm, a specialized cytoplasm, predisposes cells in Drosophila’s posterior end to develop into germ cell progenitors. Then, unexpectedly, Lehmann announced that she was moving her lab to New York University in Manhattan. Unable to follow Lehmann because of family obligations, Zamore continued his work in the lab of James Williamson, an MIT biophysicist. When Williamson accepted a position at The Scripps Research Institute in La Jolla, CA, Zamore again moved his project, this time to MIT biochemist David Bartel’s lab. “At that time, it seemed like a baroque arrangement to be accreting principle investigators as they moved. But in fact, it was the single most important professional event for me because it gave me three different perspectives on how to do science, each associated with different techniques,” says Zamore. “Now, in my lab, I have several different approaches that I use.” Investigating RNAi In 1999, Zamore accepted a position at the University of Massachusetts Medical School. He chose this job over others because of the school’s collegial atmosphere, he says. “The idea here not only is that science is fun, but that it’s more fun when you do it with people you like,” Zamore notes. During the last few months in his postdoctoral position, Zamore discovered that www.acschemicalbiolog y.o rg
he and a fellow postdoc, Tom Tuschl, shared a common interest in the emerging field of RNAi. During a group meeting Zamore presented a paper written by Andrew Fire, now at Stanford University (California) School of Medicine and University of Massachusetts Medical School’s Craig Mello that gave the first details on RNAi as observed in the nematode, Caenorhabditis elegans (2 ). “Then Tom got up and said, ‘What a coincidence, I was going to talk about RNAi too,’” Zamore says. Tuschl’s plans were to set up a system to study the newly discovered phenomenon in vitro by recapitulating RNAi in a cell extract. “We needed a Lego set for RNAi—a cell extract that reproduced the process in a test tube so we could take in apart,” says Zamore. Since a recent paper had shown that RNAi could take place in Drosophila embryos, Zamore suggested using them for their model system. The two researchers partnered to create a cell extract system to recapitulate RNAi. The results from this collaborative project were published in 1999 (3 ). “By the time I showed up at the University of Massachusetts, everyone knew I wasn’t going to work on anything but this,” he says. “RNA silencing was already my passion.” The phenomenon captured his interest for a multitude of reasons, he adds. Not only does RNAi seem to be a pivotal force for fighting viruses in plants and insects, but all eukaryotic cells use self-produced double-stranded RNA pieces, called microRNAs (miRNA), to regulate gene expression. miRNA appears to play a large role in steering development of all multi cellular organisms, making sure that development proceeds normally. “If you want to understand how organisms develop complex body patterns with many different cell types that are highly specialized, you have to understand miRNAs,” Zamore says, “especially if you want to understand how they do it in such a robust manner.”
miRNA seem to play an important role during development by selectively turning down the expression of genes. miRNA also appear to dampen the expression throughout a cell’s life of so-called junk DNA, which makes up about half of the mammalian genome. “It prevents 50 percent of the genome from monopolizing important machinery, from making proteins in a nonproductive way by silencing it. You don’t have eukaryotic life without it,” Zamore says. Zamore’s research continues to expand our mechanistic understanding of RNAi. In 2001, he and his colleagues published findings indicating that cells use adenosine triphosphate to fuel gene silencing (4 ). “It was one of the first indications we had that this wasn’t a simple, passive process, but an active, ordered pathway of building an RNAi machine to do RNAi,” Zamore says. The same study revealed that cells seem to have a quality control process that prevents energy from being wasted on constructing needless RNAi machinery. “To our surprise, siRNAs that lacked 5´ phosphates didn’t get incorporated into RNAi machinery,” he adds. “Cells seem to ask a question: Are these double-stranded RNAs really for RNAi? If they’re junk, then cells don’t want to build the machinery.” In 2003, his group published an explanation for how the RNAi machinery chooses one strand of siRNA over the other to use as a template for knocking down gene expression (5 ). Their findings suggest that thermodynamic features embedded in one strand of siRNA, but not the other, mark it for entry into the RNA-induced silencing complex (RISC). “siRNAs seem to have a lot of information embedded in them,” says Zamore. “We discovered very rapidly that we could predetermine which strand is incorporated into RISC.” Similar thermo dynamic information seems to accomplish the same selective process for miRNA, the paper added, chalking up another similarity between the two gene silencing pathways. VOL.1 NO. 3 • 126—128 • 2 0 0 6
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"It was fun because we had to think hard about what kinds of stereospecific modifications might affect how the strands interact with RISC."
In a more recent paper, published in 2005, Zamore’s team investigated what happens immediately after the RNAi machinery decides which strand of the duplex siRNA to use and which to discard. The prevailing view has been that an unknown ATP-dependent helicase then unwound the two strands before the passenger strand was destroyed. However, Zamore and his colleagues propose that, instead, the core enzyme of RISC, Argonaute2, cleaves the soonto-be-discarded passenger strand during RISC loading (6 ). The key to these new findings, Zamore notes, was his team’s ability to create siRNA that contained a single, non-natural phosphodiester bond. “It was fun because we had to think hard about what kinds of stereo specific modifications might affect how the strands interact with RISC,” he says. Targeting disease Zamore’s RNAi work also extends outside the university. In 2000, he, Tuschl, and Bartel, along with Phil Sharp of MIT and Paul Schimmel of The Scripps Research Institute, founded Alnylam Pharmaceuticals, based in Cambridge, MA. Zamore currently serves on the company’s scientific advisory board. Alnylam’s mission is to find new ways to treat human diseases using RNAi. Zamore notes that gene silencing could offer a fresh opportunity to effectively treat many currently incurable ailments, ranging from cystic fibrosis to avian flu. “In theory, you can turn off nearly any gene with expression that leads to disease, whether it’s a human or viral gene, a mutant gene or just a wild-type gene that’s expressed at too high a level or in the wrong place or the wrong time,” he says. Recently, Zamore adds, Alnylam published findings demonstrating that an RNAi-derived therapeutic could knock down an endogenous, clinically relevant gene in primates (7 ). The newly developed drug targets a gene that codes for the protein 1 28
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apolipoprotein B (apoB), which metabolizes cholesterol. When investigators delivered the therapeutic agent to cynomolgus monkeys, they found that it silenced apoB mRNA by as much as 90 percent, which in turn lowered circulating LDL cholesterol concentrations by more than 80 percent. With every new experiment, Zamore and his colleagues find that new questions arise about the mechanism of RNAi and chart the course for further mechanistic studies on RNAi-mediated gene silencing. With such uncharted territory spreading out before him, Zamore adds that he expects to be steeped in RNAi for the long haul. “If it remains fun, that’s all that really matters. It has to be fun, or I won’t do the work,” he says. “I think we’ll be at it for awhile.” —Christen Brownlee, Science Writer
REFERENCES 1. Zamore, P. D., Patton, J. G, and Green, M. R. (1992) Cloning and domain structure of the mammalian splicing factor U2AF, Nature 355, 609–614. 2. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391, 806–811. 3. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P., and Sharp, P. A. (1999) Targeted mRNA degradation by double-stranded RNA in vitro, Genes Dev 13, 3191–3197. 4. Nykänen, A., Haley, B., and Zamore, P. D. (2001) ATP Requirements and small interfering RNA structure in the RNA interference pathway, Cell 107, 309–321. 5. Schwarz, D. S., Hutvágner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P. D. (2003) Asymmetry in the assembly of the RNAi enzyme complex, Cell 115, 199–208. 6. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P., and Zamore, P.D. (2005) Passenger-strand cleavage facilitates assembly of siRNA into ago2-containing RNAi enzyme complexes, Cell 123, 607–620. 7. Zimmerman, T. S., Lee, A. C. H., Akinc, A. Bramlage, B., Bumcrot, D., Fedoruk, M. N., Harborth, J., Heyes, J. A., Jeffs, L. B., John, M., Judge, A. D., Lam, K., McClintock, K., Nechev, L. V., Palmer, L. R., Racie, T., Röhl, I., Seiffert, S., Shanmugam, S., Sood, V., Soutschek, J., Toudjarska, I., Wheat, A. J., Yaworski, E., Zedalis, W., Koteliansky, V., Manoharan, M., Vornlocher, H. P., and MacLachlan, I. (2006) RNAmediated gene silencing in non-human primates, Nature, published online Mar 26, http://dx.doi. org/10.1038/nature04688.
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