RNA interference/Citable Version

In living cells, genes are expressed via a messenger RNA (mRNA) which participates in the synthesis of proteins by determining the order of the amino acid building blocks that make up the protein polymer. In turn, the catalytic activities of proteins decide many of the properties of a cell, that is, its outward phenotype.

RNA interference (RNAi) is a mechanism in eukaryotic cells that is triggered when the cells are exposed to certain double-stranded ribose nucleic acid (dsRNA) polymeric molecules. This process has been detected in many organisms, including animal, plant and protist cells. The distinguishing characteristic of RNAi is destruction of messenger RNA (mRNA) molecules sharing at least some of the sequence of dsRNA trigger molecules to which the cells have been exposed.

RNA is a polymer of nucleotide bases that is involved in carrying information stored in genes, as a DNA code, to regions of the cell involved in synthesis of protein molecules, and with using that information for assembly of accurately constructed proteins. The concepts involved in flow of information from DNA to mRNA to proteins - a process known as gene expression (see figure)- are helpful for understanding RNAi, as RNAi potently inhibits this information flow, and thus effectively silences gene expression.

In biological research RNAi is a major technological breakthrough, akin to PCR in importance. In experiments using RNAi in the fly Drosophila or in the worm Caenorhabditis elegans, the effect of the loss of function of every known gene on a molecular pathway, cellular structure, or organism phenotype can now be determined rapidly and easily.

Discovery of unexpected new coordinating roles for RNA

A C. elegans worm larva unable to shed its old cuticle after RNA interference of the gene acn-1. From: Ewer J, How the ecdysozoan changed its coat PLoS Biol 3(10) e349 doi:10.1371/journal.pbio.0030349

The revolutionary finding of RNAi resulted from the unexpected outcome of experiments by plant scientists in the USA and the Netherlands. The scientists were trying to produce petunia plants with improved flower colors by introducing extra copies of a gene encoding an enzyme for flower pigmentation into petunia plants. Surprisingly, many plants with extra copies of this gene did not show the expected deep purple or deep red flowers, but had fully white or partially white flowers (see figure). When the scientists looked more closely, they found that both the endogenous and the newly introduced transgenes, had been 'turned off'. (The term transgene refers to genes introduced into a plant by genetic manipulation from another species). Accordingly, the phenomenon was first called "co-suppression of gene expression" but the molecular mechanism was unknown.

Early Examples of Gene Silencing in Transgenic Plants. TGS: Normally when plants harboring separate transgenes encoding resistance to kanamycin (KAN) and hygromycin (HYG) are crossed, 50% of the progeny are resistant to the individual antibiotics and 25% are resistant to 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 left and middle provided by Jan Kooter and those on the right by Natalie Doetsch and Rich Jorgensen)

Figure from [1] Matzke MA, Matzke AJM Planting the Seeds of a New Paradigm PLoS Biol 2(5) e133 doi:10.1371/journal.pbio.0020133

A few years later, plant virologists made a similar observation in research aimed at increasing plants' resistance to viruses. They knew that plants expressing virus-specific proteins could show enhanced tolerance, or even resistance, to viral infection, but to their surprise they found that plants carrying only short regions of viral RNA sequences not coding for any viral protein, had the same effect. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from 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 “virus-induced gene silencing” (“VIGS”). These phenomena are collectively called post transcriptional gene silencing.

After these observations in plants, many laboratories began to look for 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 hereto inexplicable phenomenon. In October 2006, Fire and Mello won the Nobel Prize for Medicine for their discoveries on gene silencing by RNAi.

RNAi is but one of a group of mechanistically related gene silencing phenomena which have in common the existence in cells of small RNA molecules, often derived from longer dsRNA precursors, or from double-stranded regions of hair-pin shaped single-stranded RNA molecules. In these "RNA silencing" mechanisms, small RNAs silence gene expression by guiding protein complexes to their target genes or target mRNAs but vary from one other, for instance in which other cell components are involved and whether or DNA or mRNA is the main target for silencing. RNA silencing pathways exist in animals, plants, protists, and fungi. Despite their confusing variety in different organisms, many gene silencing responses share common mechanistic features, suggesting a common origin of RNA silencing early in evolution.

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

Biological origins

The RNAi pathway is thought to play a role in the defenses 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 parasitic or selfish DNA such as 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.

The native cellular RNAi machinery is used by both plants and animals to regulate groups of tens or even hundreds of cellular genes via certain key regulatory genes that produce natural substrates for the Dicer enzyme that is involved in the RNAi response. These natural Dicer substrates are called primary-micro RNAs (miRNAs). By 2006, thousands of miRNAs had been identified in plants and animals, including more than 470 in humans. For miRNA's, certain parts of the genome are transcribed into relatively short single-stranded RNA molecules that fold back on themselves in a hairpin shape to create a region with double stranded 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 C. 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 regulating 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.

Gene knockdown

RNAi has recently been used 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, by RNAi, it often causes a 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. As RNAi may not totally abolish expression of the gene, this is sometimes referred as a "knockdown", to distinguish it from "knockout" 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 C. elegans and the fruit fly Drosophila melanogaster, both common 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 might 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 might 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 used RNAi to silence the expression of the human immunodeficiency virus (HIV) in mice.

Role in plant science

RNAi 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.

More practically, 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.

Cellular and molecular mechanisms

Short RNAs derived from Dicer cleavage of dsRNA are incorporated into multi-protein effector complexes, such as RISC and RITS (RNA-induced initiation of TGS) to target mRNA degradation (RNAi/PTGS), translation inhibition, or TGS and genome modifications. Argonaute (AGO) proteins bind short RNAs and ‘shepherd’ them to appropriate effector complexes. siRNAs originate from perfect RNA duplexes, which can be produced by cell-encoded RNA-dependent RNA polymerase (RDR) activity on ssRNA templates; miRNAs originate from imperfect RNA hairpins 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. Genome modifications (DNA and histone methylation) can be targeted by short RNAs that base-pair to DNA or to nascent RNA synthesized from the target gene. Target nucleic acids are in blue, short RNAs in red, proteins and enzyme complexes as ovals. From: Matzke MA, Matzke AJM Planting the seeds of a new paradigm PLoS Biol 2(5) e133 doi:10.1371/journal.pbio.0020133

One molecule of the Dicer protein from the single celled protist Giardia intestinalis, which catalyzes the cleavage of dsRNA to siRNAs. The ribonuclease (RNAase) enzyme domains are colored green, the PAZ domain yellow, the platform domain red, and the connector helix blue. The distance between the RNase and PAZ domains, determined by the length and angle of the connector helix, has been suggested as the determinant for the length of siRNA molecules produced by a given Dicer variant.(After Wikipedia figure caption in the RNAi article).

Double-stranded regions of RNA are the triggers for RNAi

There are two types of triggers for RNAi. These (1) fully double stranded RNA (dsRNA, and (2) 'hairpin' forms of single-stranded RNA in which the 'stem' of the RNA hairpin structure has complementary RNA base-pairing between two different parts of the same RNA strand (see figure to left). These two types of trigger structure can both be substrates for a ribonuclease enzyme that is appropriately named 'Dicer'.

Dicer

The RNAi process requires active participation of cellular machinery, and the properties of the Dicer enzyme are important for understanding how this process occurs.

Dicer has two active sites, both able to cleave its substrate RNAs. These active sites are separated some distance from one another in the Dicer enzyme (see figure to the right ) and this separation explains the size of the RNA fragments they produce when they act on a double stranded RNA (dsRNA) target, a dicing process which is an early step in the cellular pathway that brings about RNAi.

Multi-protein transcriptional silencing complexes

Dicer binds to and cleaves short double-stranded trigger RNA molecules (dsRNA) in two positions 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 when it acts on dsRNA are called small interfering RNAs (siRNAs), or miRNA when it acts of the cell's own transcribed, hairpin-forming miRNA precursors. One RNA strand from these siRNAs is generated by an aggregate of several proteins, including DICER, called the RNA-induced silencing complex (RISC).

Cellular RNA-dependent RNA polymerase can also produce double-stranded triggers

In plants, protozoa, fungi, and nematodes, a cell-encoded RNA-dependent RNA polymerase (cRdRP, RDR; see figures) produces fully dsRNA trigger molecules for RNA from single-stranded RNA. The structure of cell-encoded RNA-dependent RNA polymerase from Neurospora crassa (shown in the figure below) is relatively compact dimeric molecule and its core is a catalytic apparatus and protein folding strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription.

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: Jones R RNA silencing sheds light on the RNA world PLoS Biol 4(12) e448 doi:10.1371/journal.pbio.0040448

Argonautes are Slicers that cleave mRNA with a little help from their friends

The previously mentioned RISC complex has an important role in processing trigger molecules to generate short single stranded effectors of interference, and RISC complexes contain both DICER and an Argonaute protein. The human RISC complex shows a nearly 10-fold greater activity using the pre-miRNA Dicer substrate over duplex siRNA. RISC is able distinguishes the guide strand of the siRNA from the passenger strand, and specifically incorporates only the guide strand.

After integration into the RISC, single guide strands of RNA, either from siRNAs or miRNAs, 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. Proteins with similar sequence to Argonaute (e.g. RDE-1, P-element associated wimpy testes (Piwi)) are present in nearly every eukaryote, from fungi to plants, flies, and mammals, often as gene families .

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 because of the absence of RNA replicase in these organisms. In plants, RNAi is thought to propagate through cells via the transfer of siRNAs through plasmodesmata.

References

Further reading

• Ahlquist P (2002) RNA-dependent RNA polymerases, viruses, and RNA silencing Science 296:1270-73
• Baulcombe D (2004) RNA silencing in plants Nature 431:356–63
• Cogoni C, Macino G (1999) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase Nature 399:166–9
• Lippman Z, Martienssen R (2004) The role of RNA interference in heterochromatic silencing. Nature 431:364–70.
• Makeyev EV, Bamford DH (2002) Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. Mol Cell 10:1417–27
• Matzke M et al (2001) RNA: guiding gene silencing Science 293, 1080-3
• Mello CC, Conte D Jr (2004) Revealing the world of RNA interference Nature 431:338–42
• Sharp PA (2001) RNA interference--2001 Genes Dev 15:485-90
• Sijen T et al (2001) On the role of RNA amplification in dsRNA-triggered gene silencing Cell 107:465–76

See also

• The central dogma
• RNA silencing
• 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.