SCIENCE & TECHNOLOGY
RNA INTERFERENCE THE HOW AND WHY Scientists work to figure out the mechanisms underpinning gene-silencing technique CELIA M. HENRY, C&EN WASHINGTON
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N RECENT YEARS, SCIENTISTS HAVE
found that RNA—once thought to be little more than a bit player in the pathway from gene to protein —stands more center stage, playing a larger and more varied biological role than previously thought. One of these roles is in the process known as RNA interference, or RNAi. RNAi has generated much excitement within the life sciences community because it allows biologists to do genetic studies that were previously difficult. Biologists have a much better understanding of the final result of RNAi than they do of how the process itself works. They know in "a big picture sort of way" that RNAi does indeed result in reduced gene expression, but they have only just begun to understand the phenomena at a mechanistic level. In RNAi, pieces of double-stranded RNA that are 21 to 23 nucleotides long, known as short interfering RNA (siRNA), are incorporated into a protein complex called the RNA-induced silencing complex (RISC). The complex cleaves messenger RNA that is complementary to the siRNA, resulting in reduced expression of a target gene. In essence, RNAi allows biologists to "knock down" gene expression to see how the absence of that gene affects the organism without having to breed "knockouts" that lack the gene. Among the puzzles surrounding RNAi is that only one of the strands from the RNA duplex is incorporated into RISC. What characteristics determine which strand is picked? For an siRNA to be functional against a target mRNA, the antisense strand must be the one that makes it into RISC, and even then, there's no guarantee it will actually silence the gene. Scientists would like to know how they could ensure that
the antisense strand is the chosen one. (The antisense strand is the noncoding strand that is complementary to the strand coding for a gene.) For biologists who are interested in using RNAi as a tool to study genetics in organisms where such direct studies used to be difficult, if not impossible, it's enough just to know that RNAi works. Other scientists are interested in understanding the process itself— how and why it works—so that they can improve it. Two such scientists are Phillip D. Zamore, associate professor of biochemistry and molecular pharmacology at the University of Massachusetts Med-
ical School, in Worcester, and Anastasia Khvorova, vice president of research at Dharmacon in Lafayette, Colo. Zamore
and Khvorova (in t h e group led by Sumedhajayasena at Amgen) independently took different approaches to arrive at the same conclusion: T h e stability (both absolute and relative) at the 5' ends of the two siRNA strands determines which one enters RISC [Cell, 115, 199 and 2 0 9 (2003)}. The strand with the less stable 5' end is more likely to be incorporated in RISC because it is easier to unwind. T h e researchers both presented their work at a meeting on the cell biology of RNAi held at the National Academy of Sciences in May. "IT'S BECOMING increasingly clear from our work and complementary work by several other labs that the single most common defect in siRNAs [occurs when} the sense strand ends up in RISC," causing the siRNA to be considered nonfunctional, Zamore explains. "In fact, those types of siRNAs are perfectly functional. It's just that they're not functional against the desired target. My hope is that, with these new insights into the machinery itself, people will be able to avoid that pitfall." One way to tell which strand will be incorporated into RISC is to look at the "internal stability profile" for the strands, Khvorova says. The internal stability is a thermodynamic measurement that describes how tightly the two R N A strands are held together. Rather than being a single value for the entire duplex, it varies along the length of the strand. It is a property of each individual strand but only in the context of the duplex. "We realized that when you look at internal stability profiles of functional versus nonfunctional siRNAs, they are drastically different," Khvorova says. The main differences are at the 5' ends of the strands and the base pairs at positions 11-14, with the antisense strand being less stable in functional siRNAs. Dharmacon chemically modifies the sense strands of siRNAs to block their entry into RISC. In about 30% of the cases, they were able to substantially improve the functionality of the siRNAs by modifying the sense strands, Khvorova says. She won't identify the exact nature of the modifications,
Biologists have a much better understanding of the final result of RNAi than they do of how the process itself works. 16
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Richard can research but says that they can be on the backbone, the ribose, or the base. It turns out, howev er, that inefficient entry of the antisense strand into RISC is only part of the problem. "It ap pears that siRNA activ ity on average is limited by inefficient RISC en try in only 3 0 % of cas es," Khvorova says. "Ύου can take 100 nonfunc tional siRNAs. You alter the thermodynamics ei ther by chemical modi fications or structural elements, and you fully recover the activity of on ly 30 of those siRNAs. Seventy are still not fully active. You will get effi cient entry into RISC, but they are not func tional because they are not able to work in the later stages [of the RNAi pathway]. "The research ers don't yet know what other aspects of siRNAs affect their functionality
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tisense strand, making t h a t end easier to un wind. He says that by us ing this approach, it's even possible to resur rect "dead" siRNAs that incorporate t h e sense strand or neither strand. Now that they think they understand the fac tors that promote effi cient incorporation in RISC, Zamore and his colleagues are moving on to try to develop a mechanistic u n d e r s t a n d i n g of other aspects of the RNAi pathway "Part of the groundwork for un derstanding [the RNAi mechanism] is being able to describe the R N A i pathway in terms of clas sical enzymology," he says. He and his colleagues recently published a ki netic analysis of the RNAi enzyme complex [Nat. Struct. Mol Biol., 11, 599 (2004)}.The analysis sug gests that the different re gions of the siRNA play distinct roles in the cat alytic cycle. The 3' end of the siRNA seems not to contribute to the binding of the complex to the tar get mRNA. However, it's also clear that 3' comple mentarity is important for siRNA function. "When there's mismatch at the 3' end of the siRNA with its target, cleavage is slower," Zamore says. "It was sur prising to me how cleanly one could separate con tributions to binding from contributions to catalytic function."
Zamore uses the term "functional asymmetry" to describe the tenden cy of one strand of the siRNA to be preferen tially incorporated into CYCLING A mature RISC. "We can take any RNA-induced silencing sequence, including those complex incorporating that start out highly sym the correct short metric," meaning that the interfering RNA (siRNA) strands have a close-tostrand recognizes, equal likelihood of being cleaves, and releases its incorporated into RISC, target mRNA before "and convert it to one going back to do it again. that's functionally asym The 3' (hydroxyl) and 5' metric," he says. (phosphate) ends of the Zamore's strategy for siRNA are indicated. introducing functional asymmetry turns out to There is also evidence be quite simple. "We simply change a sin that siRNA is involved in other biologi gle base pair on the sense strand. We usu cal processes such as the formation of ally change position 19 [counting from heterochromatin, the D N A packing ma the 5' end] so that it's now a mismatch terial found in parts of the chromosomes with the first position of the antisense where there are few genes. An under strand." (Although both strands are 21 standing of how siRNA and RNAi work nucleotides long, they each have a twowill help illuminate those areas of biolo nucleotide overhang, so the strands share gy as well. only 19 base pairs.) "It doesn't seem to "It's already clear that siRNA has very matter what the mismatch is, just that deep biological tentacles," Zamore says. it's a mismatch," he says. This mismatch "The relationship of the mechanism to the helps destabilize the 5' end of the anbiology is a very interesting problem." • H T T P : / / W W W . C E N - O N L I N E . ORG
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