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 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 Caenorhabditis elegans.
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 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 not all details of this mechanism are known, it appears that the machinery, once it finds a double-stranded RNA molecule, cuts it up with an enzyme known as Dicer, 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 life cycle and replication of many viruses involves a double-stranded RNA stage, 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 330 microRNAs have been detected in humans.
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 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. A. Fire and C. Mello at Stanford and UMass Worcester respectively, injected double stranded RNA into C. elegans and noticed a potent gene silencing effect (Fire et al., 1998). They coined the term RNAi.Gene knockdown
RNAi has recently been applied as an experimental technique to "knockout" genes in model organisms for experimental analysis in determining 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 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 HIV virus in mice. Clinical trials performed on human HIV patients began the fall of 2005.References