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RNA interference
RNA interference (RNAi) is a molecular mechanism in which fragments of double-stranded ribonucleic acid (dsRNA) interfere with the expression of a particular gene that shares a homologous sequence (an identical stretch) with the dsRNA.
In 2006, Americans Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference.
Before RNA interference was well characterized, the phenomenon was known by other names, including post transcriptional gene silencing and transgene silencing. Only after these phenomena were also characterized at the molecular level was it obvious that they were one and the same phenomenon.
RNA interference is distinct and different from other gene-silencing phenomena in that the gene silencing caused by RNAi can spread from cell to cell and generate heritable phenotypes in first-generation progeny when used in the roundworm Caenorhabditis elegans.
Well before RNAi was discovered, RNA was used to reduce gene expression in plant genetics. Single-stranded antisense RNA was introduced into plant cells and hybridized to the homologous single-stranded "sense" messenger RNA. It is now clear that the resulting dsRNA was responsible for reducing gene expression.
Overview
RNAi has become a widespread research tool and therapeutic lead. Its initial description in animals 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[1] 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. [2] More specifically, Fire and Mello investigated the regulation of muscle protein production (in worms) using different forms of RNA. Both mRNA and an antisense RNA sequence had no effect on protein production. However, double-stranded RNA (sense & antisense together) successfully silenced the gene. Mello later wrote a review speaking of the 'RNA Revelation': "RNA has always been in control of the cell, not DNA".[citation needed] 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 treatments 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).[3]
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. [4] 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.[3]
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.[5] The pathway is conserved across all eukaryotes, although it has been independently recruited to play other functions such as histone modification,[6] the reorganization of genomic regions with complementary sequence to induce heterochromatin formation,[7] and maintenance of centromeric heterochromatin.[8]
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.[9] 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.[10] 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.[11]
History
The revolutionary finding of RNAi resulted from the unexpected outcome of experiments performed by plant scientists in the USA and The Netherlands.[12] 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[13]. 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.[14] 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.[15]
Role in medicine
It may be possible to exploit the RNA interference process for therapeutic purposes. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful.[16] The first applications to reach clinical trials are in the treatment of macular degeneration and respiratory syncytial virus.[17] RNAi has also been shown effective in the complete reversal of induced liver failure in mouse models.[18]
Other proposed clinical uses explored in cell culture center on antiviral therapies, including the inhibition of viral gene expression in cancerous cells,[19] the silencing of hepatitis A[20] and hepatitis B[21] genes, silencing of influenza gene expression,[22] and inhibition of measles viral replication.[23] Potential treatments for neurodegenerative diseases have also been proposed, with particular attention to the polyglutamine diseases such as Huntington's disease.[24]
Despite the proliferation of promising cell culture studies for RNAi-based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for "off-target" effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed.[25] A computational genomics study estimated that the error rate of off-target interactions is about 10%.[26] One major study of liver disease in mice led to high death rates in the experimental animals, suggested by researchers to be the result of "oversaturation" of the dsRNA pathway. |
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