RNA interference/Citable Version

RNA interference (RNAi) is a process triggered when cells are exposed to certain double-stranded RNA (dsRNA) molecules.

The defining response that is generated in cells undergoing RNAi is destruction of a messenger RNA (mRNA) sharing at least some of the sequence of the dsRNA trigger.

RNAi, is but one of a group of mechanistically related gene silencing phenomena which have in common small RNAs, often derived from longer dsRNA precursors, that silence gene expression by guiding protein complexes to their targeted genes or mRNAs.

Such RNA silencing pathways exist in animals, plants, protists, fungi and even bacteria. In eukaryotes, be they plant, animal or microorganism, many of these gene silencing responses share common mechanistic features, most likely reflecting the common origin of RNA-silencing early in evolution.

The existence of these ancient mechanisms were first clearly recognised in the 1980s, when plant scientists experimenting with transgenic petunias observed strange losses of gene expression in transgenic plants which were extremly difficult to explain by the models of gene regulation current at that time .

Early Examples of Gene Silencing in Transgenic Plants. TGS: Normally when two plants harboring separate transgenes encoding resistance to kanamycin (kan) or hygromycin (hyg), respectively, are crossed, 50% of the progeny are resistant to the individual antibiotics and 25% are resistant to a combination of both (top). In cases of silencing, expression of the KAN marker is extinguished in the presence of the HYG marker, as indicated by only 25% kan resistance and no double resistance (middle). PTGS: Transformation of wild-type petunia (bottom left) with a transgene encoding a pigment protein can lead to loss of pigment (white areas) owing to cosuppression of the transgene and homologous endogenous plant gene. (Photos on the left and in the middle were provided by Jan Kooter and on the right were provided by Natalie Doetsch and Rich Jorgensen.) From: Planting the Seeds of a New Paradigm Matzke MA, Matzke AJM PLoS Biology Vol. 2, No. 5, e133 doi:10.1371/journal.pbio.0020133

Research biologist's interest in RNAi really exploded when a key 1998 article Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans was published by Andrew Fire, Craig Mello and others (for which Fire and Mello were awarded a Nobel Prize in 2006). This paper described some very surprising effects of injection of dsRNA into the roundworm 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.

It has been realised that both plants and animals use RNA silencing to regulate groups (tens to hundreds) of genes by generating small single stranded RNAs (microRNA or miRNA) from transcripts of their own genes which form haipin structures containing double-stranded regions. These miRNAs regulated cell behaviour by triggering mechanistically similar responses to those occuring during RNAi.

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).

Cellular mechanism

Short RNAs derived from Dicer cleavage of dsRNA are incorporated into multiprotein effector complexes, such as RISC and RITS (RNA-induced initiation of TGS) (Verdel et al. 2004) to target mRNA degradation (RNAi/PTGS), translation inhibition, or TGS and genome modifications. ARGONAUTE (AGO) proteins (the name comes from a plant mutant [Bohmert et al. 1998]) bind short RNAs and ‘shepherd’ them to appropriate effector complexes (Carmell et al. 2002). siRNAs originate from perfect RNA duplexes, which can be produced by RDR activity on ssRNA templates; miRNAs originate from imperfect RNA hairpins that are encoded in intergenic regions of plant and animal genomes. Functions are shown at the bottom. In addition to roles in transgene silencing, both TGS and RNAi/PTGS control genome parasites called transposons (Flavell 1994; Plasterk 2002). Genome modifications (DNA and histone methylation) can potentially be targeted by short RNAs that basepair to DNA or to nascent RNA synthesized from the target gene (Grewal and Moazed 2003). Target nucleic acids are shown in blue, short RNAs in red, proteins and enzyme complexes as ovals.From: Planting the Seeds of a New Paradigm Matzke MA, Matzke AJM PLoS Biology Vol. 2, No. 5, e133 doi:10.1371/journal.pbio.0020133

RNAi is a gene silencing process that requires active participation of cellular machinery. Although the mechanism is poorly understood, it is known that the ribonuclease enzyme Dicer binds to and cleaves short double-stranded trigger 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).. In plants, protozoa, fungi, and nematodes, a cell-encoded RNA-dependent RNA polymerase (cRdRP) produces double-stranded RNA trigger molecules from single-stranded RNA. The structure cRdRP from Neurospora crassa is relatively compact dimeric molecule and its core is a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription.

The native cellular purpose of the RNAi machinery is not well characterized, but it is 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.

Schematic showing the fold of the QDE-1 RNA interference polymerase. The dimeric molecule is shown with the polypeptide chains colored from blue at the N termini to red at the C termini. From: RNA Silencing Sheds Light on the RNA World Jones R PLoS Biology Vol. 4, No. 12, e448 doi:10.1371/journal.pbio.0040448

Biological origins

The RNA interference pathway is thought to play a role in the defences against viruses and other foreign genetic material, both in animals (as part of the immune response) but especially in plants where it may 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 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. In October 2006 Fire and Mello won the Nobel Prize for Medicine for their discoveries on gene silencing by RNA intereference.

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.

Role in plant science

RNA interference is now widely used to probe gene functions in plant science. An example is a recent study from the laboratory of Jorge Dubcovsky where it was necessary to determine the function of a gene GPC-B1 that was thought to be involved in regulating wheat leaf senescence (and to affect cereal protein content) . This laboratory used RNAi to knockdown expression of the GPC-B1 gene in cereal wheat and found spectacular changes in grain germination after knockdown. The GPC-B1 knockdown wheat variety showed 30% less grain protein, zinc and iron, without differences in grain size, and verified that a single gene was responsible for all the effects. They suggest that increased expression of the gene cam improve grain protein content and nutritional value.

In a more practical vein, Australia's CSIRO has developed a new experimental wheat variety with the potential to provide benefits in the areas of bowel health, diabetes and obesity.In this case RNAi was used in wheat to increase the content of amylose, a form of starch that is more resistant to digestion .

CSIRO and others have argued that cisgenic plants - that is plants created by genetic manipulation using RNAi (which they dub "GM-lite")- pose less risks than addition of genes from other species (transgenics) as (they argue) new proteins are unlikely to be produced.

References

Further reading

• Ahlquist, P. (2002). RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing. Science 296, 1270-1273.
• Baulcombe D (2004) RNA silencing in plants. Nature 431: 356–363.
• Cogoni C, Macino G (1999) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399: 166–169.
• Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293: 1146–1150. Lippman Z, Martienssen R (2004) The role of RNA interference in heterochromatic silencing. Nature 431: 364–370.
• Makeyev EV, Bamford DH (2002) Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. Mol Cell 10: 1417–1427.
• Matzke, M., Matzke, A. J., and Kooter, J. M. (2001). RNA: guiding gene silencing. Science 293, 1080-1083.
• Mello CC, Conte D Jr (2004) Revealing the world of RNA interference. Nature 431 338–342.
• Sharp, P. A. (2001). RNA interference--2001. Genes Dev. 15, 485-490.
• Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, et al. (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107: 465–476.
• Tijsterman, M., Ketting, R. F., and Plasterk, R. H. (2002). The genetics of RNA silencing. Annu. Rev. Genet. 36, 489-519.
• Zamore, Phillip D. (2006) Essay: RNA Interference: Big Applause for Silencing in Stockholm Cell Volume 127, Issue 6, 15 December 2006, Pages 1083-1086 doi:10.1016/j.cell.2006.12.001

See also

• Small interfering RNA
• micro RNA
• Biotechnology and Plant breeding

External links

• Matzke MA, Matzke AJM (2004) Planting the Seeds of a New Paradigm. PLoS Biol 2(5): e133 DOI: 10.1371/journal.pbio.0020133, a PLoS primer on plant biologists and the understanding of RNAi
• Animation of the RNAi process, from Nature
• 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
• The Genetics of RNA silencing.