Roundtable Review

The Nobel for RNAi: From petunias to potential cure
Posted 14 June '13
800px-RNAi

RNAi is a process in which small RNA molecules activate a cellular response able to destroy a specific mRNA. Author: Richard Robinson. Under License: Creative Commons Attribution 2.5 Generic. Source: http://commons.wikimedia.org/wiki/File:RNAi.jpg

In 1986, a plant scientist failed in his attempt to produce intensely purple petunias and instead ignited a revolution in the world of genetics. Unwittingly, his experiment had demonstrated RNA interference (RNAi). This fundamental cellular mechanism regulates which of our genes are switched on or off, therefore controlling when and where proteins are produced. Today, RNAi continues to shape research conducted in labs around the world, both as an incredibly powerful molecular tool and as a topic of interest for potential applications in medicine. To appreciate why the discovery of RNAi ranks amongst the greatest scientific breakthroughs since the structure of DNA was revealed 60 years ago, it is necessary to understand the key pathway it acts upon. The central dogma of biology dictates that the manufacture of proteins requires the transmission of a code, which contains genetic instructions, from DNA in the cell nucleus, to an intermediary messenger RNA (mRNA) molecule. The mRNA carries this precious code to the cytosol, where machinery responsible for translating the code, the ribosome, is located and the resulting protein is synthesised. RNAi challenges this dogma; rather than acting as a faithful mediator between genes and protein, RNA can be a spanner in the works by interfering with the flow of genetic information. When Richard Jorgensen  of the University of Arizona in Tucson tried to cultivate petunia flowers with extra purple pigment, he used a seemingly logical approach, and injected an additional pigment-producing gene to the plant. To his great surprise, the resulting flowers blossomed with two-tone or totally white petals. Jorgensen had stumbled across a curious phenomenon, which he termed ‘co-suppression’, but was later redefined as RNAi. Instead of complementing each other, the pigment genes were thwarting each other’s attempts to work properly. Jorgensen’s finding fuelled interest in this area, and a few years later, geneticists found themselves grappling with a new process termed ‘antisense’ technology, in which genes were turned off using RNA. Similar to DNA, RNA molecules contain a string of chemical bases. ‘Sense’ RNA carries the same base sequence as mRNA, whilst ‘antisense’ RNA carries a sequence complementary to mRNA. Antisense technology is based upon the theory that injecting antisense RNA into a cell stops a specific protein from being made, so effectively switches the gene off, also known as silencing. When molecular biologists Andrew Fire, working at the Carnegie Institution in Washington and Craig Mello from the  University of Massachusetts Medical School, turned their attention to this conundrum, they used Caenorhabditis elegans, a microscopic worm popular amongst researchers for its ease of use. Sense and antisense RNAs were injected separately into worms and as expected, sense RNA made little impact on the production of the target protein, but surprisingly, antisense RNA also had a limited effect. However, when sense and antisense RNA were injected together, they observed a significant reduction in the target protein level. As so often happens when scientists strive towards a major breakthrough, an unexpected finding turns their research on its head: double-stranded RNA (dsRNA) molecules silence genes with the same code as itself. This dsRNA was actually a contaminant formed from antisense and sense RNA wrapping around each other. Surprisingly, the dsRNA mixture silenced the target gene ten times more efficiently than either strand in isolation. In terms of silencing potency, the dsRNA was truly greater than the sum of its parts. Even more astonishingly, RNAi was demonstrated to spread between cells, and be inherited by future generations. Fire and Mello’s monumental results were published in the journal Nature in 1998. They were rewarded for their efforts with a Nobel Prize in 2006, but more significantly, their stunning discovery heralded a new era in biological research. So how does this fascinating mechanism work? The RNAi machinery is activated when dsRNA is formed. These dsRNA molecules are chopped into fragments by a large protein complex called Dicer. Another key player, the RNA-induced silencing complex (RISC) then binds to one of the strands in the RNA fragments, allowing it to search for similar or identical mRNA. When this mRNA is detected, it pairs with the RNA fragment bound to RISC, and is cleaved and degraded. By shooting the messenger (RNA), protein cannot be produced, and the gene is effectively switched off. In many organisms, including humans, RNAi serves to regulate gene expression, controlling precisely which genes (out of a staggering 30,000 in our genome) are switched on or off. Pseudogenes, originally thought to have no function, are now known to code for micro RNA (miRNA) molecules. These miRNA molecules activate the RNAi machinery and block protein synthesis, vital in directing organism development and controlling a plethora of cellular functions. Fascinatingly, RNAi is also an important cellular weapon in the fight against viral infection. Many viruses use dsRNA for their genetic code, and when infecting a cell they inject their own toxic dsRNA. This dsRNA is immediately hunted out by Dicer, RISC is activated, and the potentially dangerous viral RNA is skillfully destroyed before the cell can succumb to infection. Today, RNAi is a widely used tool in thousands of labs, offering a highly effective way to turn off a desired gene. This allows the effect of this ‘loss-of-function’ or gene knockdown on cell morphology, protein expression, molecular pathways, and a host of other information to be determined. Reliable and efficient, application of RNAi has become central to many research areas, simplifying experiments, and saving scientists hours of hard graft. Currently, huge sums of money are being ploughed into investigations focused on the medical applications of RNAi. Silencing RNA has been shown to switch off a gene causing high blood cholesterol levels in animals, and it is hoped that RNAi may be used in the future to switch off the genetic mutations, which cause diseases such as Huntington’s, rheumatoid arthritis, and cancer. An attempt to change the colour of a petunia inadvertently changed the landscape of biological research. By unraveling the mystery of RNAi, Fire and Mello unearthed an elegant mechanism of conceptual and biological importance, and revealed one of Nature’s best kept genetic secrets.

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