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RNA interference (RNAi) is a mechanism in molecular biology where the presence of certain fragments of double-stranded ribonucleic acid (dsRNA) interferes with the expression of a particular gene which shares a homologous sequence with the dsRNA. RNAi is distinct from other gene silencing phenomena in that silencing can spread from cell to cell and generate heritable phenotypes in first generation progeny when used in the roundworm Caenorhabditis elegans.

In 2006, Americans Andrew Fire and Craig C. Mello won the Nobel Prize in Physiology or Medicine for their work on RNA interference.

Before RNAi was well characterized, it was called by other names, including post transcriptional gene silencing and transgene silencing. Only after these phenomena were characterized at the molecular level was it obvious that they were the same phenomenon.

The use of RNA to reduce expression in plants has been a common procedure for many years. Single-stranded antisense RNA was introduced into plant cells that hybridized to the cognate, single-stranded, sense messenger RNA. While scientists first believed that the resulting dsRNA helix could not be translated into a protein, it is now clear that the dsRNA triggered the RNAi response. The use of dsRNA became more widespread after the discovery of the RNAi machinery, first in petunias and later in roundworms (C. elegans).

Overview

RNAi has become a widespread research tool and therapeutic lead. Its initial description occurred in 1998 in a paper by Craig C. Mello and Andrew Fire based on research conducted with their colleagues (SiQun Xu, Mary Montgomery, Stephen Kostas, Sam Driver) at the Carnegie Institution of Washington. Published in the journal Nature, the paper detailed how tiny snippets of RNA fool the cell into destroying the gene's messenger RNA (mRNA) before it can produce a protein - effectively shutting specific genes down. Since that publication, RNAi has been recognized as an underlying process in animals, plants and lower organisms.

This ability to dramatically reduce an individual protein inside of cells makes RNAi a valuable research tool. What was once a laborious process is now as easy as sneaking an RNA molecule into the cell with a sequence that matches the RNA a researcher wants destroyed. That ability to one-by-one shut down the production of a given protein in a cell opens up previously impossible areas of research.

For example, if researchers want to know all the genes involved in a particular cellular event, they can use an RNAi library to individually disrupt each gene from making its protein, then look for the ones that interfere with the event in question.

Another area where scientists have high hopes for RNAi is in developing new RNA-based treatements for disease including a possible therapeutic role for RNAi in gene therapy.

Cellular mechanism

RNAi is a gene silencing process that requires active participation of cellular machinery. Although the specific mechanism is poorly understood, it is known that the ribonuclease enzyme Dicer binds to and cleaves short double-stranded RNA molecules (dsRNA) to produce double-stranded fragments of 21-23 base pairs with two-base single-stranded overhangs on each end. The short double-stranded fragments produced by Dicer, called small interfering RNAs (siRNAs), are then separated, presumably by an enzyme with helicase activity, and integrated into a multiprotein complex called the RNA-induced silencing complex (RISC).

The native cellular purpose of the RNA interference machinery is not well characterized, but it is known to be involved in microRNA (miRNAs) processing and the resulting translational repression. MicroRNAs, which are encoded in the genome and have a role in gene regulation, typically have incomplete base pairing and only inhibit the translation of the target mRNA; by contrast, RNA interference as used in the laboratory typically involves perfectly base-paired dsRNA molecules that induce mRNA cleavage. After integration into the RISC, siRNAs base pair to their target mRNA and induce the RISC component protein argonaute to cleave the mRNA, thereby preventing it from being used as a translation template.

Organisms vary in their cells' ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference are both systemic and heritable in plants and in C. elegans, although not in Drosophila or mammals due to the absence of RNA replicase in these organisms. In plants, RNAi is thought to propagate through cells via the transfer of siRNAs through plasmodesmata.

Biological origins

The RNA interference pathway is thought to play a role in the immune response to viruses and other foreign genetic material, especially in plants where it may also protect against the self-propagation of transposons. The pathway is conserved across all eukaryotes, although it has been independently recruited to play other functions such as histone modification, the reorganization of genomic regions with complementary sequence to induce heterochromatin formation, and maintenance of centromeric heterochromatin.

For miRNA's, certain parts of the genome are transcribed into short RNA molecules that fold back on themselves in a hairpin shape to create a double strand primary miRNA structure (pri-miRNA). The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. If base-pairing with the target is perfect or near-perfect this may result in cleavage of messenger RNA (mRNA). This is quite similar to the siRNA function, however, many miRNA's will base pair with mRNA with an imperfect match. In such cases, the miRNA causes the inhibition of translation and prevents normal function. Consequently, the RNAi machinery is important to regulate endogenous gene activity. This effect was first described for the worm Caenorhabditis elegans in 1993 by R. C. Lee et al. of Harvard University. In plants, this mechanism was first shown in the "JAW microRNA" of Arabidopsis; it is involved in the regulation of several genes that control the plant's shape. Genes have been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding an mRNA by base pairing, however they are not generally considered to be miRNA's because the Dicer enzyme is not involved.

History

The revolutionary finding of RNAi resulted from the unexpected outcome of experiments performed by plant scientists in the USA and The Netherlands. The goal was to produce petunia plants with improved flower colors. To achieve this goal, they introduced additional copies of a gene encoding a key enzyme for flower pigmentation into petunia plants. Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple or deep red flowers but carried fully white or partially white flowers. When the scientists had a closer look they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. Because of this observation the phenomenon was first named "co-suppression of gene expression" but the molecular mechanism remained unknown.

A few years later plant virologists made a similar observation. In their research they aimed towards improvement of resistance of plants against plant viruses. At that time it was known that plants expressing virus-specific proteins show enhanced tolerance or even resistance against virus infection. However, they also made the surprising observation that plants carrying only short regions of viral RNA sequences not coding for any viral protein showed the same effect. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from multiplying and spreading throughout the plant. They did the reverse experiment and put short pieces of plant gene sequences into plant viruses. Indeed, after infection of plants with these modified viruses the expression of the targeted plant gene was suppressed. They called this phenomenon “virus-induced gene silencing” or simply “VIGS”. These phenomena are collectively called post transcriptional gene silencing.

After these initial observations in plants many laboratories around the world searched for the occurrence of this phenomenon in other organisms. In 1998, Andrew Z. Fire and Craig C. Mello (at the Carnegie Institution of Washington and the University of Massachusetts Cancer Center respectively) reported a potent gene silencing effect after injecting double stranded RNA into C. elegans. They coined the term RNAi. The discovery of RNAi in C. elegans is particularly notable, as it represented the first identification of the causative agent (double stranded RNA) of this heretofore inexplicable phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work.

Gene knockdown

RNAi has recently been applied as an experimental technique to study the function of genes in model organisms. Double-stranded RNA for a gene of interest is introduced into a cell or organism, where it through RNAi causes an often drastic decrease in production of the protein the gene codes for. Studying the effects of this decrease can yield insights into the protein's role and function. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown", to distinguish it from "knockout" procedures in which expression of a gene is entirely eliminated by removing or destroying its DNA sequence.

Most functional genomics applications of RNAi have used the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, both commonly used model organisms in genetics research. C. elegans is particularly useful for RNAi research because the effects of the gene silencing are generally heritable and because delivery of the dsRNA is exceptionally easy. Via a mechanism whose details are poorly understood, bacteria such as E coli that carry the desired dsRNA can be fed to the worms and will transfer their RNA payload to the worm via the intestinal tract. This "delivery by feeding" yields essentially the same magnitude of gene silencing as do more costly and time-consuming traditional delivery methods, such as soaking the worms in dsRNA solution and injecting dsRNA into the gonads.

Role in medicine

The dsRNAs that trigger RNAi may be usable as drugs. The first application to reach clinical trials is in the treatment of macular degeneration. RNAi has also been shown effective in the complete reversal of induced liver failure in mouse models.

Another speculative use of dsRNA is in the repression of essential genes in eukaryotic human pathogens or viruses that are dissimilar from any human genes; this would be analogous to how existing drugs work.

RNAi interferes with the translation process of gene expression and appears not to interact with the DNA itself. Proponents of therapies based on RNAi suggest that the lack of interaction with DNA may alleviate some patients' concerns about alteration of their DNA (as practiced in gene therapy), and suggest that this method of treatment would likely be no more feared than taking any prescription drug. For this reason RNAi and therapies based on RNAi have attracted much interest in the pharmaceutical and biotech industries.

More recently, RNAi researchers have managed to use RNAi to silence the expression of the human immunodeficiency virus (HIV) in mice.

References

1. "Nobel prize for genetic discovery". BBC. 2006-10-02. Retrieved 2006-10-02.
2. ^ Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J. (2004). Molecular Cell Biology 5th ed. WH Freeman: New York, NY.
3. Schramke V, Allshire R. (2003). Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science 301(5636):1069-74. PMID 12869699
4. Stram Y, Kuzntzova L. (2006). Inhibition of viruses by RNA interference. Virus Genes 32(3):299-306.
5. Cerutti H, Casas-Mollano JA. (2006). On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50(2):81-99.
6. Holmquist GP, Ashley T. (2006). Chromosome organization and chromatin modification: influence on genome function and evolution. Cytogenet Genome Res 114(2):96-125.
7. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA. (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297(5588):1833-7. PMID 12193640
8. Lee RC, Feinbaum RL, Ambros V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843-54.
9. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D. (2003). Control of leaf morphogenesis by microRNAs. Nature 425(6955):257-63.
10. Morita T, Mochizuki Y, Aiba H. (2006). Translational repression is sufficient for gene silencing by bacterial small noncoding RNAs in the absence of mRNA destruction. Proc Natl Acad Sci 103(13):4858-63.
11. Napoli C., Lemieux C., and Jorgensen R. (1990) "Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans". Plant Cell 2: 279-289.
12. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature 391: 806-11
13. Dzitoyeva S, Dimitrijevic N, Manev H. (2003). Gamma-aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in Drosophila: adult RNA interference and pharmacological evidence. Proc Natl Acad Sci 100(9):5485-90. PMID 12692303
14. Fortunato A, Fraser AG. (2005). Uncover genetic interactions in Caenorhabditis elegans by RNA interference. Biosci Rep 25(5-6):299-307. PMID 16307378

External links

• Planting the Seeds of a New Paradigm, a PLoS primer on plant biologists and the understanding of RNAi
• Animation of the RNAi process, from Nature
• RNA Interference Portal
• siRNA Database
• NOVA scienceNOW explains RNAi - A 15 minute video of the Nova broadcast that aired on PBS, July 26, 2005
• RNA interference (RNAi) Database

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• RNA
• Gene expression
• Molecular genetics