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Rapid-Fire siRNA Sequencing
In 2002, RNA came out from under DNA's shadow with the discovery that a class of molecules called small RNAs control significant aspects of a gene's behavior; instead of coding for proteins, they control the transcription and translation of the RNAs that do. More recently, advances in sequencing have revealed an unexpected and remarkable diversity in these small RNAs (now called siRNAs) "In plants and mammals, depending on the tissue you look at, there are populations that literally comprise hundreds of thousands of small RNA sequences," explains Gregory Hannon, PhD, a professor at the Cold Spring Harbor Laboratory in New York and a noted RNA expert.
One of the key scientific questions posed by these exciting revelations: how to gather enough sequence information from these very complex populations to allow patterns to emerge, and potentially begin testing the biological function of these siRNAs?
Until recently, it was a slow, laborious, and expensive process, involving conventional
sequencing. "We were sequencing a few hundred small RNAs at a time," says Hannon.
Then, about two years ago, he heard from a colleague in genomics that a company
called 454 Life Sciences was developing a new sequencing system. He immediately
arranged a visit to 454's facilities to view the new Genome Sequencer 20, well
before it became commercially available.
When sequencing DNA, the Genome Sequencer 20 can sequence more than 20 million bases per 4.5-hour instrument run. System-specific software enables mapping or de novo assembly for whole-genome shotgun sequencing of genomes up to 50 megabases. But Hannon realized that DNA sequencing wasn't the only thing the system could do. "I immediately had about a million ideas as to how we could use this technology, and small RNA sequencing was the first one. We've now used 454's technology to move from sequencing a few hundred small RNAs to a few hundred thousand," he says.
That exponential leap in amount of information that is generated has yielded important discoveries already. "With these populations that are so complex, if you map just a few hundred of these to the genome, they just appear as a relatively scattered pattern," he says. "But when you map hundreds of thousands, it becomes clear that at least in the mammalian germline, there are loci that emerge that give rise to many thousands of individual different small RNAs. We've found that there are probably 20 loci in human, mouse, or rat genomes that act as sources for these small RNAs, and that wouldn't have been apparent from the much more limited sequencing possible with earlier technologies."
Hannon and colleagues Anglique Girard, Ravi Sachidanandam, and Michelle A. Carmell recently published their first paper on the results of their sequencing efforts. In the July 13 edition of Nature, they reported that a previously uncharacterized germline-specific class of small RNAs binds mammalian Piwi proteins. They've dubbed the class Piwi-interacting RNAs (piRNAs).
Perhaps most surprising in a field where unexpected bumps in the scientific road are common, Hannon has encountered virtually no problems in utilizing the system for his research. "It's worked like a charm from the start," he says. "The only problems that I have are getting enough money to do this, and the turnaround time from 454, as they're very highly subscribed right now. It takes us about a month to get the data but of course, the volume of data that this produces is so significant that we're immediately overwhelmed once we get it. The time it takes for turnaround is easily dwarfed by the time it takes to understand what we've learned and verify it."
The Genome Sequencer 20 technology isn't cheap, Hannon notes, but it's much less expensive than previous options for sequencing small RNAs and yields infinitely more information. "I don't think we could have done before, at any price, what we can do now. It's essentially paradigm shifting."
Gregory Hannon
Gregory Hannon joined the faculty of the Cold Spring Harbor Laboratory in 1996.
Now a professor in Cold Spring Harbor's Watson School of Biological Sciences,
he focuses his research on determining the mechanism of RNA interference (RNAi),
as well as using RNAi in the study of cancer development and investigating small
interfering RNAs (siRNAs) as possible cancer therapeutic agents.
Dr. Hannon's laboratory identified the effector complex of RNAi, which is called RISC, and showed that it contained small RNAs, now known as siRNAs. His discovery of the Dicer enzyme, an RNAseIII family member that cleaves dsRNAs into discretely sized small RNAs that enter RISC, revealed the origin of such small RNAs. He also identified another enzyme at the heart of the RNAi mechanism: argonaute2 (“Slicer”), the central enzyme in targeted cleavage of messenger RNA by siRNAs.
Dr Hannon’s research was recognized by Science magazine in 2002 as the Breakthrough of the Year and in 2005 by Esquire magazine’s “Genius Issue” as a Breakthrough of the Decade. He received his B.A. in Biochemistry and Ph.D. in Molecular Biology from Case Western Reserve University. He was a 1997 Pew Scholar in Biomedical Sciences and won a U.S. Army Breast Cancer Research Program Innovator Award and the 2005 American Association for Cancer Research Award for Outstanding Achievement in Cancer Research. In 2005, he was named a Howard Hughes Medical Institute Investigator.
Conservation of piRNA clusters among mammalian species. a, piRNAs cloned from a region of synteny between mouse, rat and human. Red, positive strand; green, negative strand. b, Genome-wide representation of the synteny of clusters. The entire genome of each species is represented vertically with chromosome 1 at the top and the sex chromosomes at the bottom. Grey lines indicate synteny. Only clusters consisting of more than 1,000 piRNAs, or showing synteny to clusters of more than 1,000 piRNAs, are depicted.
Reprinted by permission from Macmillan Publishers Ltd: Nature, 442:7099(199-202), copyright 2006.
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