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RNA interference

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RNA interference (RNAi) is a mechanism in molecular biology where the presence of certain fragments of double-stranded RNA (dsRNA) interferes with the expression of a particular gene.

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.

In plants, the usage of double stranded RNA called antisense mRNA to reduce expression has been a common procedure for many years. The use of antisense RNA became more widespread after the discovery of the RNAi machinery, first in petunias and later in roundworms (C. elegans).

Overview

RNAi appears to be a highly potent and specific process which is actively carried out by special mechanisms in the cell, known as the RNA interference machinery. While the complete details of how it works are still unknown, it appears that the machinery, once it finds a double-stranded RNA molecule, cuts it up, separates the two strands, and then proceeds to destroy other single-stranded RNA molecules that are complementary to one of those segments. dsRNAs direct the creation of small interfering RNAs (siRNAs) which target RNA-degrading enzymes (RNAses) to destroy transcripts complementary to the siRNAs.

The genetic information of many viruses is held in the form of double-stranded RNA, so it is likely that the RNA interference machinery evolved as a defense against these viruses. The machinery is however also used by the cell itself to regulate gene activity: certain parts of the genome are transcribed into microRNA, short RNA molecules that fold back on themselves in a hairpin shape to create a double strand. When the RNA interference machinery detects these double strands, it will also destroy all mRNAs that match the microRNA, thus preventing their translation and lowering the activity of many other genes. 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. The mechanism has also been shown in many other eukaryotes; by now, some 150 microRNAs have been detected in humans.

RNAi has been linked to various cellular processes, including the formation of centromeric structure [1] and gene regulation, through microRNAs and heterochromatin formation[2].

Gene knockdown

RNAi has recently been applied as an experimental technique to "knockout" genes in model organisms for experimental analysis in determing the function of a gene. Repressing a gene from being expressed allows for testing of the protein and its role in the life of a cell or larger organism. Since RNAi may not totally abolish expression of a gene, using it against a gene is sometimes referred as a "knockdown", to distinguish it from procedures in which the DNA sequence encoding a gene is removed.

Most functional genomics applications of RNAi were made on Caenorhabditis elegans, a nematode that is frequently used as a model organism in genetics research.

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.

History

The revolutionary finding of RNAi resulted from the unexpected outcome of experiments performed by plant scientists in the USA and the Netherlands (Napoli et al., 1990). 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 phenmenona 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. A. Fire and C. Mello injected double stranded RNA into C. elegans and noticed a potent gene silencing effect (Fire et al., 1998). They coined the term RNAi,

References

  1. 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.
  2. Dehio C. and Schell J. (1994). "Identification of plant genetic loci involved in a post transcriptional mechanism for meiotically reversible transgene silencing". Proceedings of the National Academy of Sciences of the United States of America 91 (12): 5538-5542.
  3. 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

External links

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