RNA interference
Encyclopedia
RNA interference is a process within living cells that moderates the activity of their gene
s. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. In 2006, Andrew Fire
and Craig C. Mello shared the Nobel Prize in Physiology or Medicine
for their work on RNA interference in the nematode
worm C. elegans, which they published in 1998.
Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA
(siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA
(mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein
. RNA interference has an important role in defending cells against parasitic genes – virus
es and transposon
s – but also in directing development
as well as gene expression
in general.
The RNAi pathway is found in many eukaryote
s including animals and is initiated by the enzyme Dicer
, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of ~20 nucleotide
s that are called siRNAs. Each siRNA is unwound into two single-stranded (ss) ssRNAs, namely the passenger strand and the guide strand. The passenger strand will be degraded, and the guide strand is incorporated into the RNA-induced silencing complex
(RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute
, the catalytic component of the RISC complex. In some organisms this process is known to spread systemically despite the initially limited molar concentrations of siRNA.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture
and in living organisms because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division
. Exploitation of the pathway is also a promising tool in biotechnology
and medicine
.
RNAi is an RNA-dependent gene silencing
process that is controlled by the RNA-induced silencing complex
(RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm
, where they interact with the catalytic RISC component argonaute
. When the dsRNA is exogenous (coming from infection by a virus
with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by the enzyme. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding gene
s in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop
structure of pre-miRNA in the nucleus
, then exported to the cytoplasm to be cleaved by Dicer. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC complex.
protein Dicer
, which binds and cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 20–25 base pair
s with a 2-nucleotide overhang at the 3' end. Bioinformatics
studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects. These short double-stranded fragments are called small interfering RNAs (siRNA
s). These siRNAs are then separated into single strands and integrated into an active RISC complex. After integration into the RISC, siRNAs base-pair to their target mRNA and induce cleavage of the mRNA, thereby preventing it from being used as a translation
template.
Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans
and R2D2 in Drosophila
, that stimulates dicer activity. This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown. This RNA-binding protein then facilitates the transfer of cleaved siRNAs to the RISC complex.
In C. elegans
, this initiation response is amplified through the synthesis of a population of 'secondary' siRNAs during which the dicer-produced initiating or 'primary' siRNAs are used as templates. These 'secondary' siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase
(RdRP).
MicroRNAs (miRNAs) are genomically
encoded non-coding RNA
s that help regulate gene expression
, particularly during development
. The phenomenon of RNA interference, broadly defined, includes the endogenously induced gene silencing effects of miRNAs as well as silencing triggered by foreign dsRNA. Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but before reaching maturity, miRNAs must first undergo extensive post-transcriptional modification
. An miRNA is expressed from a much longer RNA-coding gene as a primary transcript
known as a pri-miRNA which is processed, in the cell nucleus
, to a 70-nucleotide stem-loop
structure called a pre-miRNA by the microprocessor complex. This complex consists of an RNase III
enzyme called Drosha
and a dsRNA-binding protein DGCR8. The dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing.
The siRNAs derived from long dsRNA precursors differ from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target. In Drosophila
and C. elegans
, miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.
The active components of an RNA-induced silencing complex
(RISC) are endonuclease
s called argonaute
proteins, which cleave the target mRNA strand complementary
to their bound siRNA. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation. Although it was first believed that an ATP
-dependent helicase
separated these two strands, the process is actually ATP-independent and performed directly by the protein components of RISC. The strand selected as the guide tends to be the one whose 5' end is less stably paired to its complement, but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation. Instead, the R2D2 protein may serve as the differentiating factor by binding the more-stable 5' end of the passenger strand.
The structural basis for binding of RNA to the argonaute protein was examined by X-ray crystallography
of the binding domain of an RNA-bound argonaute protein. Here, the phosphorylated
5' end of the RNA strand enters a conserved
basic surface pocket
and makes contacts through a divalent
cation (an atom with two positive charges) such as magnesium
and by aromatic
stacking
(a process that allows more than one atom to share an electron by passing it back and forth) between the 5' nucleotide in the siRNA and a conserved tyrosine
residue. This site is thought to form a nucleation site for the binding of the siRNA to its mRNA target.
It is not understood how the activated RISC complex locates complementary mRNAs within the cell. Although the cleavage process has been proposed to be linked to translation
, translation of the mRNA target is not essential for RNAi-mediated degradation. Indeed, RNAi may be more effective against mRNA targets that are not translated. Argonaute proteins, the catalytic components of RISC, are localized to specific regions in the cytoplasm
called P-bodies
(also cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay; miRNA activity is also clustered in P-bodies. Disruption of P-bodies decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.
Components of the RNA interference pathway are also used in many eukaryotes in the maintenance of the organization and structure of their genome
s. Modification of histone
s and associated induction of heterochromatin
formation serves to downregulate genes pre-transcriptionally
; this process is referred to as RNA-induced transcriptional silencing
(RITS), and is carried out by a complex of proteins called the RITS complex. In fission yeast this complex contains argonaute
, a chromodomain
protein Chp1, and a protein called Tas3 of unknown function. As a consequence, the induction and spread of heterochromatic regions requires the argonaute and RdRP proteins. Indeed, deletion of these genes in the fission yeast S. pombe
disrupts histone methylation
and centromere
formation, causing slow or stalled anaphase
during cell division
. In some cases, similar processes associated with histone modification have been observed to transcriptionally upregulate genes.
The mechanism by which the RITS complex induces heterochromatin formation and organization is not well understood, and most studies have focused on the mating-type region
in fission yeast, which may not be representative of activities in other genomic regions or organisms. In maintenance of existing heterochromatin regions, RITS forms a complex with siRNAs complementary
to the local genes and stably binds local methylated histones, acting co-transcriptionally to degrade any nascent pre-mRNA transcripts that are initiated by RNA polymerase
. The formation of such a heterochromatin region, though not its maintenance, is dicer-dependent, presumably because dicer is required to generate the initial complement of siRNAs that target subsequent transcripts. Heterochromatin maintenance has been suggested to function as a self-reinforcing feedback loop, as new siRNAs are formed from the occasional nascent transcripts by RdRP for incorporation into local RITS complexes. The relevance of observations from fission yeast mating-type regions and centromeres to mammal
s is not clear, as heterochromatin maintenance in mammalian cells may be independent of the components of the RNAi pathway.
that is most prevalent in higher eukaryotes converts adenosine nucleotides into inosine
in dsRNAs via the enzyme adenosine deaminase
(ADAR). It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate. Indeed, some pre-miRNAs do undergo A→I RNA editing, and this mechanism may regulate the processing and expression of mature miRNAs. Furthermore, at least one mammalian ADAR can sequester siRNAs from RNAi pathway components. Further support for this model comes from studies on ADAR-null C. elegans strains indicating that A→I RNA editing may counteract RNAi silencing of endogenous genes and transgenes.
, although not in Drosophila
or mammal
s. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata
(channels in the cell walls that enable communication and transport). The heritability comes from methylation
of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell. A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression. This translational effect may be produced by inhibiting the interactions of translation initiation factor
s with the messenger RNA's polyadenine tail
.
Some eukaryotic protozoa such as Leishmania major
and Trypanosoma cruzi
lack the RNAi pathway entirely. Most or all of the components are also missing in some fungi, most notably the model organism
Saccharomyces cerevisiae
. A recent study however reveals the presence of RNAi in other budding yeast species such as Saccharomyces castellii and Candida albicans
, further demonstrating that inducing two RNAi-related proteins from S. castellii facilitates RNAi in S. cerevisiae. That certain ascomycetes and basidiomycetes are missing RNA interference pathways indicates that proteins required for RNA silencing have been lost independently from many fungal lineages
, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantage in certain niche
s.
.
es and other foreign genetic material
, especially in plants where it may also prevent the self-propagation of transposon
s. Plants such as Arabidopsis thaliana
express multiple dicer homologs
that are specialized to react differently when the plant is exposed to different types of viruses. Even before the RNAi pathway was fully understood, it was known that induced gene silencing in plants could spread throughout the plant in a systemic effect, and could be transferred from stock to scion plants via grafting
. This phenomenon has since been recognized as a feature of the plant adaptive immune system, and allows the entire plant to respond to a virus after an initial localized encounter. In response, many plant viruses have evolved elaborate mechanisms that suppress the RNAi response in plant cells. These include viral proteins that bind short double-stranded RNA fragments with single-stranded overhang ends, such as those produced by the action of dicer. Some plant genomes also express endogenous siRNAs in response to infection by specific types of bacteria
. These effects may be part of a generalized response to pathogens that downregulates any metabolic processes in the host that aid the infection process.
Although animals generally express fewer variants of the dicer enzyme than plants, RNAi in some animals has also been shown to produce an antiviral response. In both juvenile and adult Drosophila, RNA interference is important in antiviral innate immunity and is active against pathogens such as Drosophila X virus
. A similar role in immunity may operate in C. elegans, as argonaute proteins are upregulated in response to viruses and worms that overexpress components of the RNAi pathway are resistant to viral infection.
The role of RNA interference in mammalian innate immunity is poorly understood, and relatively little data is available. However, the existence of viruses that encode genes able to suppress the RNAi response in mammalian cells may be evidence in favour of an RNAi-dependent mammalian immune response. However, this hypothesis of RNAi-mediated immunity in mammals has been challenged as poorly substantiated. Alternative functions for RNAi in mammalian viruses also exist, such as miRNAs expressed by the herpes virus
that may act as heterochromatin
organization triggers to mediate viral latency.
s, including both intron
ic and intergenic
miRNAs, are most important in translational repression
and in the regulation of development
, especially on the timing of morphogenesis
and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cell
s. The role of endogenously expressed miRNA in downregulating gene expression
was first described in C. elegans in 1993. In plants this function was discovered when the "JAW microRNA" of Arabidopsis
was shown to be involved in the regulation of several genes that control plant shape. In plants, the majority of genes regulated by miRNAs are transcription factor
s; thus miRNA activity is particularly wide-ranging and regulates entire gene networks during development by modulating the expression of key regulatory genes, including transcription factors as well as F-box protein
s. In many organisms, including humans, miRNAs have also been linked to the formation of tumor
s and dysregulation of the cell cycle
. Here, miRNAs can function as both oncogene
s and tumor suppressors.
. Part of the mechanism for how these RNA upregulate genes is known: dicer
and argonaute
are involved, possibly via histone demethylation
. miRNAs have also been proposed to upregulate their target genes upon cell cycle arrest, although the underlying mechanisms have not been elucidated.
of all eukaryote
s most likely already possessed an early RNA interference pathway; the absence of the pathway in certain eukaryotes is thought to be a derived characteristic. This ancestral RNAi system probably contained at least one dicer
-like protein, one argonaute
, one PIWI protein, and an RNA-dependent RNA polymerase
that may have also played other cellular roles. A large-scale comparative genomics
study likewise indicates that the eukaryotic crown group
already possessed these components, which may then have had closer functional associations with generalized RNA degradation systems such as the exosome
. This study also suggests that the RNA-binding argonaute protein family, which is shared among eukaryotes, most archaea, and at least some bacteria (such as Aquifex aeolicus
), is homologous to and originally evolved from components of the translation initiation
system.
The ancestral function of the RNAi system is generally agreed to have been immune defense against exogenous genetic elements such as transposon
s and viral
genomes. Related functions such as histone
modification may have already been present in the ancestor of modern eukaryotes, although other functions such as regulation of development by miRNA are thought to have evolved later.
RNA interference genes, as components of the antiviral innate immune system in many eukaryotes, are involved in an evolutionary arms race
with viral genes. Some viruses have evolved mechanisms for suppressing the RNAi response in their host cells, an effect that has been noted particularly for plant viruses. Studies of evolutionary rates in Drosophila have shown that genes in the RNAi pathway are subject to strong directional selection
and are among the fastest-evolving
genes in the Drosophila genome
.
and in vivo in model organism
s. Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects of this decrease can show the physiological role of the gene product. 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.
Extensive efforts in computational biology
have been directed toward the design of successful dsRNA reagents that maximize gene knockdown but minimize "off-target" effects. Off-target effects arise when an introduced RNA has a base sequence that can pair with and thus reduce the expression of multiple genes at a time. Such problems occur more frequently when the dsRNA contains repetitive sequences. It has been estimated from studying the genomes of H. sapiens, C. elegans
, and S. pombe that about 10% of possible siRNAs will have substantial off-target effects. A multitude of software tools have been developed implementing algorithm
s for the design of general, mammal-specific, and virus-specific siRNAs that are automatically checked for possible cross-reactivity.
Depending on the organism and experimental system, the exogenous RNA may be a long strand designed to be cleaved by dicer, or short RNAs designed to serve as siRNA substrates. In most mammal
ian cells, shorter RNAs are used because long double-stranded RNA molecules induce the mammalian interferon
response, a form of innate immunity that reacts nonspecifically to foreign genetic material. Mouse oocyte
s and cells from early mouse embryo
s lack this reaction to exogenous dsRNA and are therefore a common model system for studying gene-knockdown effects in mammals. Specialized laboratory techniques have also been developed to improve the utility of RNAi in mammalian systems by avoiding the direct introduction of siRNA, for example, by stable transfection
with a plasmid
encoding the appropriate sequence from which siRNAs can be transcribed, or by more elaborate lentiviral
vector systems allowing the inducible activation or deactivation of transcription, known as conditional RNAi.
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Most functional genomics
applications of RNAi in animals have used C. elegans and Drosophila, as these are the common model organism
s in which RNAi is most effective. C. elegans is particularly useful for RNAi research for two reasons: firstly, the effects of the gene silencing are generally heritable, and secondly because delivery of the dsRNA is extremely simple. Through 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" is just as effective at inducing gene silencing as more costly and time-consuming delivery methods, such as soaking the worms in dsRNA solution and injecting dsRNA into the gonads. Although delivery is more difficult in most other organisms, efforts are also underway to undertake large-scale genomic screening applications in cell culture with mammalian cells.
Approaches to the design of genome-wide RNAi libraries can require more sophistication than the design of a single siRNA for a defined set of experimental conditions. Artificial neural network
s are frequently used to design siRNA libraries and to predict their likely efficiency at gene knockdown. Mass genomic screening is widely seen as a promising method for genome annotation and has triggered the development of high-throughput screening methods based on microarray
s. However, the utility of these screens and the ability of techniques developed on model organisms to generalize to even closely related species has been questioned, for example from C. elegans to related parasitic nematodes.
Functional genomics using RNAi is a particularly attractive technique for genomic mapping and annotation in plants because many plants are polyploid, which presents substantial challenges for more traditional genetic engineering methods. For example, RNAi has been successfully used for functional genomics studies in bread wheat (which is hexaploid) as well as more common plant model systems Arabidopsis
and maize
.
It may be possible to exploit RNA interference in therapy. 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. Among the first applications to reach clinical trial
s were in the treatment of macular degeneration
and respiratory syncytial virus, RNAi has also been shown to be effective in the reversal of induced liver failure in mouse models.
Other proposed clinical uses center on antiviral therapies, including topical microbicide treatments that use RNAi to treat infection (at Harvard University Medical School; in mice, so far) by herpes simplex virus
type 2 and the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and coreceptors for HIV
, the silencing of hepatitis A
and hepatitis B genes, silencing of influenza
gene expression, and inhibition of measles
viral replication. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease
. RNA interference is also often seen as a promising way to treat cancer
by silencing genes differentially upregulated in tumor
cells or genes involved in cell division
. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector
systems similar to those suggested for gene therapy
.
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. A computational genomics study estimated that the error rate of off-target interactions is about 10%. 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, due to the use of shRNAs that have to be processed in the nucleus and exported to the cytoplasm using an active mechanism. All these are considerations that are under active investigation, to reduce their impact in the potential therapeutic applications for RNAi.
RNA interference-based applications are being developed to target persistent HIV-1 infection. Viruses like HIV-1 are particularly difficult targets for RNAi-attack because they are escape-prone, which requires combinatorial RNAi strategies to prevent viral escape. The future of antiviral RNAi therapeutics is very promising, but it remains of critical importance to include many controls in pre-clinical test models to unequivocally demonstrate sequence-specific action of the RNAi inducers.
, particularly in the engineering of food plants that produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton
seeds are rich in dietary protein
but naturally contain the toxic terpenoid
product gossypol
, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase
, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pests. Similar efforts have been directed toward the reduction of the cyanogenic
natural product linamarin
in cassava
plants.
Although no plant products that use RNAi-based genetic engineering
have yet passed the experimental stage, development efforts have successfully reduced the levels of allergen
s in tomato
plants and decreased the precursors of likely carcinogen
s in tobacco
plants. Other plant traits that have been engineered in the laboratory include the production of non-narcotic
natural products by the opium poppy
, resistance to common plant viruses, and fortification of plants such as tomatoes with dietary antioxidant
s. Previous commercial products, including the Flavr Savr tomato and two cultivar
s of ringspot
-resistant papaya
, were originally developed using antisense technology but likely exploited the RNAi pathway.
(HTS) technology. The RNAi HTS technology allows genome-wide loss-of-function screening and is broadly used in the identification of genes associated with specific biological phenotypes. This technology has been hailed as the second genomics wave, following the first genomics wave of gene expression
microarray
and single nucleotide polymorphism
discovery platforms
.
One of the major advantages of the genome-scale RNAi screening is its ability to simultaneously interrogate thousands of genes. With the ability of generating a large amount of data per experiment, the genome-scale RNAi screening has led to an explosion in the rate of generating data. Consequently, one of the most fundamental challenges in the genome-scale RNAi researches is to glean biological significance from mounds of data, which requires the adoption of suitable statistics/bioinformatics methods such as those presented in a recently published book
for analyzing RNAi screens. The basic process of cell-based RNAi screening includes (i) the choice of an RNAi library, (ii) the selection of a robust and stable type of cells, (iii) the transfection of the selected cells with RNAi agents from the chosen RNAi library, (iv) necessary treatment or incubation, (v) signal detection, (vi) statistical and bioinformatics analysis, and (vii) the determination of important genes or therapeutical targets
.
The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants, and more directly by reports of unexpected outcomes in experiments performed by plant scientists in the United States
and the Netherlands
in the early 1990s. In an attempt to alter flower
colors in petunia
s, researchers introduced additional copies of a gene encoding chalcone synthase
, a key enzyme for flower pigmentation into petunia plants of normally pink or violet flower color. The overexpressed gene was expected to result in darker flowers, but instead produced less pigmented, fully or partially white flowers, indicating that the activity of chalcone synthase had been substantially decreased; in fact, both the endogenous genes and the transgenes were downregulated in the white flowers. Soon after, a related event termed quelling was noted in the fungus
Neurospora crassa
, although it was not immediately recognized as related. Further investigation of the phenomenon in plants indicated that the downregulation was due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation. This phenomenon was called co-suppression of gene expression, but the molecular mechanism remained unknown.
Not long after, plant virologists working on improving plant resistance to viral diseases observed a similar unexpected phenomenon. While it was known that plants expressing virus-specific proteins showed enhanced tolerance or resistance to viral infection, it was not expected that plants carrying only short, non-coding regions of viral RNA sequences would show similar levels of protection. Researchers believed that viral RNA produced by transgenes could also inhibit viral replication. The reverse experiment, in which short sequences of plant genes were introduced into viruses, showed that the targeted gene was suppressed in an infected plant. This phenomenon was labeled "virus-induced gene silencing" (VIGS), and the set of such phenomena were 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. Craig C. Mello and Andrew Fire
's 1998 Nature paper reported a potent gene silencing effect after injecting double stranded RNA into C. elegans
. In investigating the regulation of muscle protein production, they observed that neither mRNA nor antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term RNAi. Fire and Mello's discovery was particularly notable because it represented the first identification of the causative agent for the phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine
in 2006 for their work.
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. In 2006, Andrew Fire
Andrew Fire
Andrew Zachary Fire is an American biologist and professor of pathology and of genetics at the Stanford University School of Medicine. He was awarded the 2006 Nobel Prize for Physiology or Medicine, along with Craig C. Mello, for the discovery of RNA interference...
and Craig C. Mello shared the Nobel Prize in Physiology or Medicine
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine administered by the Nobel Foundation, is awarded once a year for outstanding discoveries in the field of life science and medicine. It is one of five Nobel Prizes established in 1895 by Swedish chemist Alfred Nobel, the inventor of dynamite, in his will...
for their work on RNA interference in the nematode
Nematode
The nematodes or roundworms are the most diverse phylum of pseudocoelomates, and one of the most diverse of all animals. Nematode species are very difficult to distinguish; over 28,000 have been described, of which over 16,000 are parasitic. It has been estimated that the total number of nematode...
worm C. elegans, which they published in 1998.
Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA
Small interfering RNA
Small interfering RNA , sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference pathway, where it...
(siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
(mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
. RNA interference has an important role in defending cells against parasitic genes – virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es and transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
s – but also in directing development
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
as well as gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
in general.
The RNAi pathway is found in many eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s including animals and is initiated by the enzyme Dicer
Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments called small interfering RNA about 20-25 nucleotides long, usually with a two-base overhang on the 3' end...
, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of ~20 nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s that are called siRNAs. Each siRNA is unwound into two single-stranded (ss) ssRNAs, namely the passenger strand and the guide strand. The passenger strand will be degraded, and the guide strand is incorporated into the RNA-induced silencing complex
RNA-induced silencing complex
RNA-Induced Silencing Complex, or RISC, is a multiprotein complex that incorporates one strand of a small interfering RNA or micro RNA . RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA...
(RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
, the catalytic component of the RISC complex. In some organisms this process is known to spread systemically despite the initially limited molar concentrations of siRNA.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
and in living organisms because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
. Exploitation of the pathway is also a promising tool in biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
and medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
.
Cellular mechanism
Gene silencing
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification...
process that is controlled by the RNA-induced silencing complex
RNA-induced silencing complex
RNA-Induced Silencing Complex, or RISC, is a multiprotein complex that incorporates one strand of a small interfering RNA or micro RNA . RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA...
(RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
, where they interact with the catalytic RISC component argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
. When the dsRNA is exogenous (coming from infection by a virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by the enzyme. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding gene
Non-coding RNA
A non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
s in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop
Stem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...
structure of pre-miRNA in the nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
, then exported to the cytoplasm to be cleaved by Dicer. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC complex.
dsRNA cleavage
Endogenous dsRNA initiates RNAi by activating the ribonucleaseRibonuclease
Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.-Function:All organisms studied contain...
protein Dicer
Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments called small interfering RNA about 20-25 nucleotides long, usually with a two-base overhang on the 3' end...
, which binds and cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 20–25 base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
s with a 2-nucleotide overhang at the 3' end. Bioinformatics
Bioinformatics
Bioinformatics is the application of computer science and information technology to the field of biology and medicine. Bioinformatics deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, software...
studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects. These short double-stranded fragments are called small interfering RNAs (siRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
s). These siRNAs are then separated into single strands and integrated into an active RISC complex. After integration into the RISC, siRNAs base-pair to their target mRNA and induce cleavage of the mRNA, thereby preventing it from being used as a translation
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
template.
Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
and R2D2 in Drosophila
Drosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
, that stimulates dicer activity. This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown. This RNA-binding protein then facilitates the transfer of cleaved siRNAs to the RISC complex.
In C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, this initiation response is amplified through the synthesis of a population of 'secondary' siRNAs during which the dicer-produced initiating or 'primary' siRNAs are used as templates. These 'secondary' siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase
RNA-dependent RNA polymerase
RNA-dependent RNA polymerase , , or RNA replicase, is an enzyme that catalyzes the replication of RNA from an RNA template...
(RdRP).
MicroRNA
MicroRNAs (miRNAs) are genomically
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
encoded non-coding RNA
Non-coding RNA
A non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
s that help regulate gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
, particularly during development
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
. The phenomenon of RNA interference, broadly defined, includes the endogenously induced gene silencing effects of miRNAs as well as silencing triggered by foreign dsRNA. Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but before reaching maturity, miRNAs must first undergo extensive post-transcriptional modification
Post-transcriptional modification
Post-transcriptional modification is a process in cell biology by which, in eukaryotic cells, primary transcript RNA is converted into mature RNA. A notable example is the conversion of precursor messenger RNA into mature messenger RNA , which includes splicing and occurs prior to protein synthesis...
. An miRNA is expressed from a much longer RNA-coding gene as a primary transcript
Primary transcript
A primary transcript is an RNA molecule that has not yet undergone any modification after its synthesis. For example, a precursor messenger RNA is a primary transcript that becomes a messenger RNA after processing, and a primary microRNA precursor becomes a microRNA after processing....
known as a pri-miRNA which is processed, in the cell nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
, to a 70-nucleotide stem-loop
Stem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...
structure called a pre-miRNA by the microprocessor complex. This complex consists of an RNase III
RNase III
RNase III enzymes specifically bind to and cleave double-stranded RNA . There are three subdivisions, known as Class 1, 2, and 3.Prokaryotic ribonuclease III is an enzyme that digests double-stranded RNA. It is involved in the processing of ribosomal RNA precursors and of some mRNAs.* Class 1...
enzyme called Drosha
Drosha
Drosha is a Class 2 RNase III enzyme responsible for initiating the processing of microRNA , or short RNA molecules naturally expressed by the cell that regulate a wide variety of other genes by interacting with the RNA-induced silencing complex to induce cleavage of complementary messenger RNA ...
and a dsRNA-binding protein DGCR8. The dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing.
The siRNAs derived from long dsRNA precursors differ from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target. In Drosophila
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
and C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.
RISC activation and catalysis
RNA-induced silencing complex
RNA-Induced Silencing Complex, or RISC, is a multiprotein complex that incorporates one strand of a small interfering RNA or micro RNA . RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA...
(RISC) are endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
s called argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
proteins, which cleave the target mRNA strand complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to their bound siRNA. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation. Although it was first believed that an ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
-dependent helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
separated these two strands, the process is actually ATP-independent and performed directly by the protein components of RISC. The strand selected as the guide tends to be the one whose 5' end is less stably paired to its complement, but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation. Instead, the R2D2 protein may serve as the differentiating factor by binding the more-stable 5' end of the passenger strand.
The structural basis for binding of RNA to the argonaute protein was examined by X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
of the binding domain of an RNA-bound argonaute protein. Here, the phosphorylated
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
5' end of the RNA strand enters a conserved
Conserved sequence
In biology, conserved sequences are similar or identical sequences that occur within nucleic acid sequences , protein sequences, protein structures or polymeric carbohydrates across species or within different molecules produced by the same organism...
basic surface pocket
Binding site
In biochemistry, a binding site is a region on a protein, DNA, or RNA to which specific other molecules and ions—in this context collectively called ligands—form a chemical bond...
and makes contacts through a divalent
Divalent
In chemistry, a divalent ion or molecule has a valence of two and thus can form two bonds with other ions or molecules. An older term for divalent is bivalent....
cation (an atom with two positive charges) such as magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
and by aromatic
Aromaticity
In organic chemistry, Aromaticity is a chemical property in which a conjugated ring of unsaturated bonds, lone pairs, or empty orbitals exhibit a stabilization stronger than would be expected by the stabilization of conjugation alone. The earliest use of the term was in an article by August...
stacking
Stacking (chemistry)
In chemistry, pi stacking refers to attractive, noncovalent interactions between aromatic rings. These interactions are historically thought to be important in to base stacking of DNA nucleotides, protein folding, template-directed synthesis, materials science, and molecular recognition, although...
(a process that allows more than one atom to share an electron by passing it back and forth) between the 5' nucleotide in the siRNA and a conserved tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
residue. This site is thought to form a nucleation site for the binding of the siRNA to its mRNA target.
It is not understood how the activated RISC complex locates complementary mRNAs within the cell. Although the cleavage process has been proposed to be linked to translation
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
, translation of the mRNA target is not essential for RNAi-mediated degradation. Indeed, RNAi may be more effective against mRNA targets that are not translated. Argonaute proteins, the catalytic components of RISC, are localized to specific regions in the cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
called P-bodies
P-bodies
Processing bodies were first described in the scientific literature by Bashkirov et. al. in 1997, in which they describe "small granules… discrete, prominent foci" as the cytoplasmic location of the mouse exoribonuclease mXrn1p. It wasn’t until 2002 that a glimpse into the nature and importance of...
(also cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay; miRNA activity is also clustered in P-bodies. Disruption of P-bodies decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.
Transcriptional silencing
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
s. Modification of histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
s and associated induction of heterochromatin
Heterochromatin
Heterochromatin is a tightly packed form of DNA, which comes in different varieties. These varieties lie on a continuum between the two extremes of constitutive and facultative heterochromatin...
formation serves to downregulate genes pre-transcriptionally
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
; this process is referred to as RNA-induced transcriptional silencing
RNA-induced transcriptional silencing
RNA-induced transcriptional silencing is a form of RNA interference by which short RNA molecules - such as small interfering RNA - trigger the downregulation of transcription of a particular gene or genomic region. This is usually accomplished by posttranslational modification of histone tails...
(RITS), and is carried out by a complex of proteins called the RITS complex. In fission yeast this complex contains argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
, a chromodomain
Chromodomain
A chromodomain is a protein structural domain of about 40-50 amino acid residues commonly found in proteins associated with the remodeling and manipulation of chromatin. The domain is highly conserved among both plants and animals, and is represented in a large number of different proteins in many...
protein Chp1, and a protein called Tas3 of unknown function. As a consequence, the induction and spread of heterochromatic regions requires the argonaute and RdRP proteins. Indeed, deletion of these genes in the fission yeast S. pombe
Schizosaccharomyces pombe
Schizosaccharomyces pombe, also called "fission yeast", is a species of yeast. It is used as a model organism in molecular and cell biology. It is a unicellular eukaryote, whose cells are rod-shaped. Cells typically measure 3 to 4 micrometres in diameter and 7 to 14 micrometres in length...
disrupts histone methylation
Histone methylation
Histone methylation is the modification of certain amino acids in a histone protein by the addition of one, two, or three methyl groups. In the cell nucleus, DNA is wound around histones...
and centromere
Centromere
A centromere is a region of DNA typically found near the middle of a chromosome where two identical sister chromatids come closest in contact. It is involved in cell division as the point of mitotic spindle attachment...
formation, causing slow or stalled anaphase
Anaphase
Anaphase, from the ancient Greek ἀνά and φάσις , is the stage of mitosis or meiosis when chromosomes move to opposite poles of the cell....
during cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
. In some cases, similar processes associated with histone modification have been observed to transcriptionally upregulate genes.
The mechanism by which the RITS complex induces heterochromatin formation and organization is not well understood, and most studies have focused on the mating-type region
Mating-type region
The mating-type region is a specialized region in the genomes of some yeast and other fungi, usually organized into heterochromatin and possessing unique histone methylation patterns. The genes in this region regulate the mating type of the organism and therefore determine key events in its life...
in fission yeast, which may not be representative of activities in other genomic regions or organisms. In maintenance of existing heterochromatin regions, RITS forms a complex with siRNAs complementary
Complementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
to the local genes and stably binds local methylated histones, acting co-transcriptionally to degrade any nascent pre-mRNA transcripts that are initiated by RNA polymerase
RNA polymerase
RNA polymerase is an enzyme that produces RNA. In cells, RNAP is needed for constructing RNA chains from DNA genes as templates, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses...
. The formation of such a heterochromatin region, though not its maintenance, is dicer-dependent, presumably because dicer is required to generate the initial complement of siRNAs that target subsequent transcripts. Heterochromatin maintenance has been suggested to function as a self-reinforcing feedback loop, as new siRNAs are formed from the occasional nascent transcripts by RdRP for incorporation into local RITS complexes. The relevance of observations from fission yeast mating-type regions and centromeres to mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
s is not clear, as heterochromatin maintenance in mammalian cells may be independent of the components of the RNAi pathway.
Crosstalk with RNA editing
The type of RNA editingRNA editing
The term RNA editing describes those molecular processes in which the information content in an RNA molecule is altered through a chemical change in the base makeup. To date, such changes have been observed in tRNA, rRNA, mRNA and microRNA molecules of eukaryotes but not prokaryotes...
that is most prevalent in higher eukaryotes converts adenosine nucleotides into inosine
Inosine
Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring via a β-N9-glycosidic bond....
in dsRNAs via the enzyme adenosine deaminase
Adenosine deaminase
Adenosine deaminase is an enzyme involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues.-Reactions:...
(ADAR). It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate. Indeed, some pre-miRNAs do undergo A→I RNA editing, and this mechanism may regulate the processing and expression of mature miRNAs. Furthermore, at least one mammalian ADAR can sequester siRNAs from RNAi pathway components. Further support for this model comes from studies on ADAR-null C. elegans strains indicating that A→I RNA editing may counteract RNAi silencing of endogenous genes and transgenes.
Variation among organisms
Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference can be both systemic and heritable in plants and C. elegansCaenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, although not in Drosophila
Drosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
or mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
s. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata
Plasmodesmata
Plasmodesmata are microscopic channels which traverse the cell walls of plant cells and some algal cells, enabling transport and communication between them. Species that have plasmodesmata include members of the Charophyceae, Charales and Coleochaetales , as well as all embryophytes, better known...
(channels in the cell walls that enable communication and transport). The heritability comes from methylation
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell. A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression. This translational effect may be produced by inhibiting the interactions of translation initiation factor
Eukaryotic initiation factor
Eukaryotic initiation factors are proteins involved in the initiation phase of eukaryotic translation. They function in forming a complex with the 40S ribosomal subunit and Met-tRNAi called the 43S preinitation complex , recognizing the 5' cap structure of mRNA and recruiting the 43S PIC to mRNA,...
s with the messenger RNA's polyadenine tail
Polyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
.
Some eukaryotic protozoa such as Leishmania major
Leishmania major
Leishmania major is a species of Leishmania.It is associated with zoonotic cutaneous leishmaniasis.The genome has been sequenced....
and Trypanosoma cruzi
Trypanosoma cruzi
Trypanosoma cruzi is a species of parasitic euglenoid trypanosomes. This species causes the trypanosomiasis diseases in humans and animals in America...
lack the RNAi pathway entirely. Most or all of the components are also missing in some fungi, most notably the model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
. A recent study however reveals the presence of RNAi in other budding yeast species such as Saccharomyces castellii and Candida albicans
Candida albicans
Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans. Systemic fungal infections including those by C...
, further demonstrating that inducing two RNAi-related proteins from S. castellii facilitates RNAi in S. cerevisiae. That certain ascomycetes and basidiomycetes are missing RNA interference pathways indicates that proteins required for RNA silencing have been lost independently from many fungal lineages
Lineage (evolution)
An evolutionary lineage is a sequence of species, that form a line of descent, each new species the direct result of speciation from an immediate ancestral species. Lineages are subsets of the evolutionary tree of life. Lineages are often determined by the techniques of molecular systematics.-...
, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantage in certain niche
Ecological niche
In ecology, a niche is a term describing the relational position of a species or population in its ecosystem to each other; e.g. a dolphin could potentially be in another ecological niche from one that travels in a different pod if the members of these pods utilize significantly different food...
s.
Related prokaryotic systems
Gene expression in prokaryotes is influenced by an RNA-based system similar in some respects to RNAi. Here, RNA-encoding genes control mRNA abundance or translation by producing a complementary RNA that binds to an mRNA by base pairing. However these regulatory RNAs are not generally considered to be analogous to miRNAs because the dicer enzyme is not involved. It has been suggested that CRISPR interference systems in prokaryotes are analogous to eukaryotic RNA interference systems, although none of the protein components are orthologousHomology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
.
Immunity
RNA interference is a vital part of the immune response to virusVirus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es and other foreign genetic material
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
, especially in plants where it may also prevent the self-propagation of transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
s. Plants such as Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
express multiple dicer homologs
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
that are specialized to react differently when the plant is exposed to different types of viruses. Even before the RNAi pathway was fully understood, it was known that induced gene silencing in plants could spread throughout the plant in a systemic effect, and could be transferred from stock to scion plants via grafting
Grafting
Grafting is a horticultural technique whereby tissues from one plant are inserted into those of another so that the two sets of vascular tissues may join together. This vascular joining is called inosculation...
. This phenomenon has since been recognized as a feature of the plant adaptive immune system, and allows the entire plant to respond to a virus after an initial localized encounter. In response, many plant viruses have evolved elaborate mechanisms that suppress the RNAi response in plant cells. These include viral proteins that bind short double-stranded RNA fragments with single-stranded overhang ends, such as those produced by the action of dicer. Some plant genomes also express endogenous siRNAs in response to infection by specific types of bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
. These effects may be part of a generalized response to pathogens that downregulates any metabolic processes in the host that aid the infection process.
Although animals generally express fewer variants of the dicer enzyme than plants, RNAi in some animals has also been shown to produce an antiviral response. In both juvenile and adult Drosophila, RNA interference is important in antiviral innate immunity and is active against pathogens such as Drosophila X virus
Drosophila X virus
Drosophila X virus is a virus that can infect fruit flies of the genus Drosophila and is commonly used to study innate immunity in the common model organism Drosophila melanogaster. The viral capsid is roughly hexagonal and icosahedrally symmetrical. The genome of the virus consists of two linear,...
. A similar role in immunity may operate in C. elegans, as argonaute proteins are upregulated in response to viruses and worms that overexpress components of the RNAi pathway are resistant to viral infection.
The role of RNA interference in mammalian innate immunity is poorly understood, and relatively little data is available. However, the existence of viruses that encode genes able to suppress the RNAi response in mammalian cells may be evidence in favour of an RNAi-dependent mammalian immune response. However, this hypothesis of RNAi-mediated immunity in mammals has been challenged as poorly substantiated. Alternative functions for RNAi in mammalian viruses also exist, such as miRNAs expressed by the herpes virus
Herpes virus
In colloquial use, herpes virus refers to the herpes simplex virus, particularly when transmitted sexually.In scientific use, herpesvirus refers to a virus of the taxonomic family herpesviridae....
that may act as heterochromatin
Heterochromatin
Heterochromatin is a tightly packed form of DNA, which comes in different varieties. These varieties lie on a continuum between the two extremes of constitutive and facultative heterochromatin...
organization triggers to mediate viral latency.
Downregulation of genes
Endogenously expressed miRNAMirna
Mirna may refer to:geographical entities* Mirna , a river in Istria, Croatia* Mirna , a river in Slovenia, tributary of the river Sava* Mirna , a settlement in the municipality of Mirna in Southeastern Sloveniapeople...
s, including both intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
ic and intergenic
Intergenic region
An Intergenic region is a stretch of DNA sequences located between clusters of genes that contain few or no genes. Occasionally some intergenic DNA acts to control genes nearby, but most of it has no currently known function...
miRNAs, are most important in translational repression
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
and in the regulation of development
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
, especially on the timing of morphogenesis
Morphogenesis
Morphogenesis , is the biological process that causes an organism to develop its shape...
and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cell
Stem cell
This article is about the cell type. For the medical therapy, see Stem Cell TreatmentsStem cells are biological cells found in all multicellular organisms, that can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells...
s. The role of endogenously expressed miRNA in downregulating gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
was first described in C. elegans in 1993. In plants this function was discovered when the "JAW microRNA" of Arabidopsis
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
was shown to be involved in the regulation of several genes that control plant shape. In plants, the majority of genes regulated by miRNAs are transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s; thus miRNA activity is particularly wide-ranging and regulates entire gene networks during development by modulating the expression of key regulatory genes, including transcription factors as well as F-box protein
F-box protein
F-box proteins are proteins containing at least one F-box domain. The first identified F-box protein is one of three components of the SCF complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome....
s. In many organisms, including humans, miRNAs have also been linked to the formation of tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
s and dysregulation of the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
. Here, miRNAs can function as both oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...
s and tumor suppressors.
Upregulation of genes
RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter can increase gene transcription, a phenomenon dubbed RNA activationRNAa
Small double-stranded RNA has been found to silence gene expression by an evolutionally conserved mechanism known as RNA interference or RNAi. Such dsRNAs are called small interfering RNAs or siRNA. RNAi can occur at both transcriptional and post-transcriptional levels...
. Part of the mechanism for how these RNA upregulate genes is known: dicer
Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments called small interfering RNA about 20-25 nucleotides long, usually with a two-base overhang on the 3' end...
and argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
are involved, possibly via histone demethylation
Histone methylation
Histone methylation is the modification of certain amino acids in a histone protein by the addition of one, two, or three methyl groups. In the cell nucleus, DNA is wound around histones...
. miRNAs have also been proposed to upregulate their target genes upon cell cycle arrest, although the underlying mechanisms have not been elucidated.
Evolution
Based on parsimony-based phylogenetic analysis, the most recent common ancestorMost recent common ancestor
In genetics, the most recent common ancestor of any set of organisms is the most recent individual from which all organisms in the group are directly descended...
of all eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s most likely already possessed an early RNA interference pathway; the absence of the pathway in certain eukaryotes is thought to be a derived characteristic. This ancestral RNAi system probably contained at least one dicer
Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments called small interfering RNA about 20-25 nucleotides long, usually with a two-base overhang on the 3' end...
-like protein, one argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
, one PIWI protein, and an RNA-dependent RNA polymerase
RNA-dependent RNA polymerase
RNA-dependent RNA polymerase , , or RNA replicase, is an enzyme that catalyzes the replication of RNA from an RNA template...
that may have also played other cellular roles. A large-scale comparative genomics
Comparative genomics
Comparative genomics is the study of the relationship of genome structure and function across different biological species or strains. Comparative genomics is an attempt to take advantage of the information provided by the signatures of selection to understand the function and evolutionary...
study likewise indicates that the eukaryotic crown group
Crown group
A crown group is a group consisting of living representatives, their ancestors back to the most recent common ancestor of that group, and all of that ancestor's descendants. The name was given by Willi Hennig, the formulator of phylogenetic systematics, as a way of classifying living organisms...
already possessed these components, which may then have had closer functional associations with generalized RNA degradation systems such as the exosome
Exosome complex
The exosome complex is a multi-protein complex capable of degrading various types of RNA molecules...
. This study also suggests that the RNA-binding argonaute protein family, which is shared among eukaryotes, most archaea, and at least some bacteria (such as Aquifex aeolicus
Aquifex aeolicus
Aquifex aeolicus is a rod-shaped prokaryote with a length of 2 to 6 micrometers and a diameter of around half a micrometer. It is one of a handful of species in the Aquificae phylum, an unusual group of thermophilic bacteria that are thought to be some of the oldest species of bacteria.A...
), is homologous to and originally evolved from components of the translation initiation
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
system.
The ancestral function of the RNAi system is generally agreed to have been immune defense against exogenous genetic elements such as transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
s and viral
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
genomes. Related functions such as histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
modification may have already been present in the ancestor of modern eukaryotes, although other functions such as regulation of development by miRNA are thought to have evolved later.
RNA interference genes, as components of the antiviral innate immune system in many eukaryotes, are involved in an evolutionary arms race
Evolutionary arms race
In evolutionary biology, an evolutionary arms race is an evolutionary struggle between competing sets of co-evolving genes that develop adaptations and counter-adaptations against each other, resembling an arms race, which are also examples of positive feedback...
with viral genes. Some viruses have evolved mechanisms for suppressing the RNAi response in their host cells, an effect that has been noted particularly for plant viruses. Studies of evolutionary rates in Drosophila have shown that genes in the RNAi pathway are subject to strong directional selection
Directional selection
In population genetics, directional selection is a mode of natural selection in which a single phenotype is favored, causing the allele frequency to continuously shift in one direction...
and are among the fastest-evolving
Evolution
Evolution is any change across successive generations in the heritable characteristics of biological populations. Evolutionary processes give rise to diversity at every level of biological organisation, including species, individual organisms and molecules such as DNA and proteins.Life on Earth...
genes in the Drosophila genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
.
Gene knockdown
The RNA interference pathway is often exploited in experimental biology to study the function of genes in cell cultureCell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
and in vivo in model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
s. Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects of this decrease can show the physiological role of the gene product. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown
Gene knockdown
Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene...
", to distinguish it from "knockout
Gene knockout
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
" procedures in which expression of a gene is entirely eliminated.
Extensive efforts in computational biology
Computational biology
Computational biology involves the development and application of data-analytical and theoretical methods, mathematical modeling and computational simulation techniques to the study of biological, behavioral, and social systems...
have been directed toward the design of successful dsRNA reagents that maximize gene knockdown but minimize "off-target" effects. Off-target effects arise when an introduced RNA has a base sequence that can pair with and thus reduce the expression of multiple genes at a time. Such problems occur more frequently when the dsRNA contains repetitive sequences. It has been estimated from studying the genomes of H. sapiens, C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, and S. pombe that about 10% of possible siRNAs will have substantial off-target effects. A multitude of software tools have been developed implementing algorithm
Algorithm
In mathematics and computer science, an algorithm is an effective method expressed as a finite list of well-defined instructions for calculating a function. Algorithms are used for calculation, data processing, and automated reasoning...
s for the design of general, mammal-specific, and virus-specific siRNAs that are automatically checked for possible cross-reactivity.
Depending on the organism and experimental system, the exogenous RNA may be a long strand designed to be cleaved by dicer, or short RNAs designed to serve as siRNA substrates. In most mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian cells, shorter RNAs are used because long double-stranded RNA molecules induce the mammalian interferon
Interferon
Interferons are proteins made and released by host cells in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.IFNs belong to...
response, a form of innate immunity that reacts nonspecifically to foreign genetic material. Mouse oocyte
Oocyte
An oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic...
s and cells from early mouse embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
s lack this reaction to exogenous dsRNA and are therefore a common model system for studying gene-knockdown effects in mammals. Specialized laboratory techniques have also been developed to improve the utility of RNAi in mammalian systems by avoiding the direct introduction of siRNA, for example, by stable transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
with a plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
encoding the appropriate sequence from which siRNAs can be transcribed, or by more elaborate lentiviral
Lentivirus
Lentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period...
vector systems allowing the inducible activation or deactivation of transcription, known as conditional RNAi.
Functional genomics
Functional genomics
Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects to describe gene functions and interactions...
applications of RNAi in animals have used C. elegans and Drosophila, as these are the common model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
s in which RNAi is most effective. C. elegans is particularly useful for RNAi research for two reasons: firstly, the effects of the gene silencing are generally heritable, and secondly because delivery of the dsRNA is extremely simple. Through a mechanism whose details are poorly understood, bacteria such as E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
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" is just as effective at inducing gene silencing as more costly and time-consuming delivery methods, such as soaking the worms in dsRNA solution and injecting dsRNA into the gonads. Although delivery is more difficult in most other organisms, efforts are also underway to undertake large-scale genomic screening applications in cell culture with mammalian cells.
Approaches to the design of genome-wide RNAi libraries can require more sophistication than the design of a single siRNA for a defined set of experimental conditions. Artificial neural network
Artificial neural network
An artificial neural network , usually called neural network , is a mathematical model or computational model that is inspired by the structure and/or functional aspects of biological neural networks. A neural network consists of an interconnected group of artificial neurons, and it processes...
s are frequently used to design siRNA libraries and to predict their likely efficiency at gene knockdown. Mass genomic screening is widely seen as a promising method for genome annotation and has triggered the development of high-throughput screening methods based on microarray
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
s. However, the utility of these screens and the ability of techniques developed on model organisms to generalize to even closely related species has been questioned, for example from C. elegans to related parasitic nematodes.
Functional genomics using RNAi is a particularly attractive technique for genomic mapping and annotation in plants because many plants are polyploid, which presents substantial challenges for more traditional genetic engineering methods. For example, RNAi has been successfully used for functional genomics studies in bread wheat (which is hexaploid) as well as more common plant model systems Arabidopsis
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
and maize
Maize
Maize known in many English-speaking countries as corn or mielie/mealie, is a grain domesticated by indigenous peoples in Mesoamerica in prehistoric times. The leafy stalk produces ears which contain seeds called kernels. Though technically a grain, maize kernels are used in cooking as a vegetable...
.
Medicine
Interferon
Interferons are proteins made and released by host cells in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.IFNs belong to...
response, the use of short interfering RNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
mimics has been more successful. Among the first applications to reach clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
s were in the treatment of macular degeneration
Macular degeneration
Age-related macular degeneration is a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field because of damage to the retina. It occurs in “dry” and “wet” forms. It is a major cause of blindness and visual impairment in older adults...
and respiratory syncytial virus, RNAi has also been shown to be effective in the reversal of induced liver failure in mouse models.
Other proposed clinical uses center on antiviral therapies, including topical microbicide treatments that use RNAi to treat infection (at Harvard University Medical School; in mice, so far) by herpes simplex virus
Herpes simplex virus
Herpes simplex virus 1 and 2 , also known as Human herpes virus 1 and 2 , are two members of the herpes virus family, Herpesviridae, that infect humans. Both HSV-1 and HSV-2 are ubiquitous and contagious...
type 2 and the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and coreceptors for HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
, the silencing of hepatitis A
Hepatitis A
Hepatitis A is an acute infectious disease of the liver caused by the hepatitis A virus , an RNA virus, usually spread the fecal-oral route; transmitted person-to-person by ingestion of contaminated food or water or through direct contact with an infectious person...
and hepatitis B genes, silencing of influenza
Influenza
Influenza, commonly referred to as the flu, is an infectious disease caused by RNA viruses of the family Orthomyxoviridae , that affects birds and mammals...
gene expression, and inhibition of measles
Measles
Measles, also known as rubeola or morbilli, is an infection of the respiratory system caused by a virus, specifically a paramyxovirus of the genus Morbillivirus. Morbilliviruses, like other paramyxoviruses, are enveloped, single-stranded, negative-sense RNA viruses...
viral replication. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease
Huntington's disease
Huntington's disease, chorea, or disorder , is a neurodegenerative genetic disorder that affects muscle coordination and leads to cognitive decline and dementia. It typically becomes noticeable in middle age. HD is the most common genetic cause of abnormal involuntary writhing movements called chorea...
. RNA interference is also often seen as a promising way to treat cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
by silencing genes differentially upregulated in tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
cells or genes involved in cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector
Viral vector
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture . Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect...
systems similar to those suggested for gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
.
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. A computational genomics study estimated that the error rate of off-target interactions is about 10%. 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, due to the use of shRNAs that have to be processed in the nucleus and exported to the cytoplasm using an active mechanism. All these are considerations that are under active investigation, to reduce their impact in the potential therapeutic applications for RNAi.
RNA interference-based applications are being developed to target persistent HIV-1 infection. Viruses like HIV-1 are particularly difficult targets for RNAi-attack because they are escape-prone, which requires combinatorial RNAi strategies to prevent viral escape. The future of antiviral RNAi therapeutics is very promising, but it remains of critical importance to include many controls in pre-clinical test models to unequivocally demonstrate sequence-specific action of the RNAi inducers.
Biotechnology
RNA interference has been used for applications in biotechnologyBiotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
, particularly in the engineering of food plants that produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton
Cotton
Cotton is a soft, fluffy staple fiber that grows in a boll, or protective capsule, around the seeds of cotton plants of the genus Gossypium. The fiber is almost pure cellulose. The botanical purpose of cotton fiber is to aid in seed dispersal....
seeds are rich in dietary protein
Protein in nutrition
Proteins are polymer chains made of amino acids linked together by peptide bonds. Proteins and carbohydrates contain 4 kcal per gram as opposed to lipids which contain 9 kcal per gram....
but naturally contain the toxic terpenoid
Terpenoid
The terpenoids , sometimes called isoprenoids, are a large and diverse class of naturally occurring organic chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in thousands of ways. Most are multicyclic structures that differ from one another not only in...
product gossypol
Gossypol
Gossypol is a natural phenol derived from the cotton plant . Gossypol is a phenolic aldehyde that permeates cells and acts as an inhibitor for several dehydrogenase enzymes. It is a yellow pigment....
, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase
Delta-cadinene synthase
In enzymology, a -delta-cadinene synthase is an enzyme that catalyzes the chemical reactionHence, this enzyme has one substrate, 2-trans,6-trans-farnesyl diphosphate, and two products, -delta-cadinene and diphosphate....
, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pests. Similar efforts have been directed toward the reduction of the cyanogenic
Cyanide
A cyanide is a chemical compound that contains the cyano group, -C≡N, which consists of a carbon atom triple-bonded to a nitrogen atom. Cyanides most commonly refer to salts of the anion CN−. Most cyanides are highly toxic....
natural product linamarin
Linamarin
Linamarin is a cyanogenic glucoside found in the leaves and roots of plants such as cassava, lima beans, and flax. It is a glucoside of acetone cyanohydrin...
in cassava
Cassava
Cassava , also called yuca or manioc, a woody shrub of the Euphorbiaceae native to South America, is extensively cultivated as an annual crop in tropical and subtropical regions for its edible starchy tuberous root, a major source of carbohydrates...
plants.
Although no plant products that use RNAi-based genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
have yet passed the experimental stage, development efforts have successfully reduced the levels of allergen
Allergen
An allergen is any substance that can cause an allergy. In technical terms, an allergen is a non-parasitic antigen capable of stimulating a type-I hypersensitivity reaction in atopic individuals....
s in tomato
Tomato
The word "tomato" may refer to the plant or the edible, typically red, fruit which it bears. Originating in South America, the tomato was spread around the world following the Spanish colonization of the Americas, and its many varieties are now widely grown, often in greenhouses in cooler...
plants and decreased the precursors of likely carcinogen
Carcinogen
A carcinogen is any substance, radionuclide, or radiation that is an agent directly involved in causing cancer. This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes...
s in tobacco
Tobacco
Tobacco is an agricultural product processed from the leaves of plants in the genus Nicotiana. It can be consumed, used as a pesticide and, in the form of nicotine tartrate, used in some medicines...
plants. Other plant traits that have been engineered in the laboratory include the production of non-narcotic
Narcotic
The term narcotic originally referred medically to any psychoactive compound with any sleep-inducing properties. In the United States of America it has since become associated with opioids, commonly morphine and heroin and their derivatives, such as hydrocodone. The term is, today, imprecisely...
natural products by the opium poppy
Opium poppy
Opium poppy, Papaver somniferum, is the species of plant from which opium and poppy seeds are extracted. Opium is the source of many opiates, including morphine , thebaine, codeine, papaverine, and noscapine...
, resistance to common plant viruses, and fortification of plants such as tomatoes with dietary antioxidant
Antioxidant
An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When...
s. Previous commercial products, including the Flavr Savr tomato and two cultivar
Cultivar
A cultivar'Cultivar has two meanings as explained under Formal definition. When used in reference to a taxon, the word does not apply to an individual plant but to all those plants sharing the unique characteristics that define the cultivar. is a plant or group of plants selected for desirable...
s of ringspot
Papaya ringspot virus
Papaya ringspot virus is a plant pathogenic virus in the genus Potyvirus and the virus family Potyviridae.The virus is a non-enveloped, flexous rod-shaped particle that is between 760-800 nm long and 12 nm in diameter. It is transmitted between plants by mechanical activities like...
-resistant papaya
Papaya
The papaya , papaw, or pawpaw is the fruit of the plant Carica papaya, the sole species in the genus Carica of the plant family Caricaceae...
, were originally developed using antisense technology but likely exploited the RNAi pathway.
Genome-scale RNAi screening
The genome-scale RNAi research relies on the high-throughput screeningHigh-throughput screening
High-throughput screening is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening allows a...
(HTS) technology. The RNAi HTS technology allows genome-wide loss-of-function screening and is broadly used in the identification of genes associated with specific biological phenotypes. This technology has been hailed as the second genomics wave, following the first genomics wave of gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
microarray
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
and single nucleotide polymorphism
Single nucleotide polymorphism
A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
discovery platforms
.
One of the major advantages of the genome-scale RNAi screening is its ability to simultaneously interrogate thousands of genes. With the ability of generating a large amount of data per experiment, the genome-scale RNAi screening has led to an explosion in the rate of generating data. Consequently, one of the most fundamental challenges in the genome-scale RNAi researches is to glean biological significance from mounds of data, which requires the adoption of suitable statistics/bioinformatics methods such as those presented in a recently published book
for analyzing RNAi screens. The basic process of cell-based RNAi screening includes (i) the choice of an RNAi library, (ii) the selection of a robust and stable type of cells, (iii) the transfection of the selected cells with RNAi agents from the chosen RNAi library, (iv) necessary treatment or incubation, (v) signal detection, (vi) statistical and bioinformatics analysis, and (vii) the determination of important genes or therapeutical targets
.
History and discovery
United States
The United States of America is a federal constitutional republic comprising fifty states and a federal district...
and the Netherlands
Netherlands
The Netherlands is a constituent country of the Kingdom of the Netherlands, located mainly in North-West Europe and with several islands in the Caribbean. Mainland Netherlands borders the North Sea to the north and west, Belgium to the south, and Germany to the east, and shares maritime borders...
in the early 1990s. In an attempt to alter flower
Flower
A flower, sometimes known as a bloom or blossom, is the reproductive structure found in flowering plants . The biological function of a flower is to effect reproduction, usually by providing a mechanism for the union of sperm with eggs...
colors in petunia
Petunia
Petunia is a widely cultivated genus of flowering plants of South American origin, closely related with tobacco, cape gooseberries, tomatoes, deadly nightshades, potatoes and chili peppers; in the family Solanaceae. The popular flower derived its name from French, which took the word petun, meaning...
s, researchers introduced additional copies of a gene encoding chalcone synthase
Chalcone synthase
Chalcone synthases are a family of polyketide synthase enzymes associated with the production of chalcones, a class of organic compounds found mainly in plants as natural defense mechanisms and as synthetic intermediates, for example in the production of pigments...
, a key enzyme for flower pigmentation into petunia plants of normally pink or violet flower color. The overexpressed gene was expected to result in darker flowers, but instead produced less pigmented, fully or partially white flowers, indicating that the activity of chalcone synthase had been substantially decreased; in fact, both the endogenous genes and the transgenes were downregulated in the white flowers. Soon after, a related event termed quelling was noted in the fungus
Fungus
A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria...
Neurospora crassa
Neurospora crassa
Neurospora crassa is a type of red bread mold of the phylum Ascomycota. The genus name, meaning "nerve spore" refers to the characteristic striations on the spores. The first published account of this fungus was from an infestation of French bakeries in 1843. N...
, although it was not immediately recognized as related. Further investigation of the phenomenon in plants indicated that the downregulation was due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation. This phenomenon was called co-suppression of gene expression, but the molecular mechanism remained unknown.
Not long after, plant virologists working on improving plant resistance to viral diseases observed a similar unexpected phenomenon. While it was known that plants expressing virus-specific proteins showed enhanced tolerance or resistance to viral infection, it was not expected that plants carrying only short, non-coding regions of viral RNA sequences would show similar levels of protection. Researchers believed that viral RNA produced by transgenes could also inhibit viral replication. The reverse experiment, in which short sequences of plant genes were introduced into viruses, showed that the targeted gene was suppressed in an infected plant. This phenomenon was labeled "virus-induced gene silencing" (VIGS), and the set of such phenomena were 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. Craig C. Mello and Andrew Fire
Andrew Fire
Andrew Zachary Fire is an American biologist and professor of pathology and of genetics at the Stanford University School of Medicine. He was awarded the 2006 Nobel Prize for Physiology or Medicine, along with Craig C. Mello, for the discovery of RNA interference...
's 1998 Nature paper reported a potent gene silencing effect after injecting double stranded RNA into C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
. In investigating the regulation of muscle protein production, they observed that neither mRNA nor antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term RNAi. Fire and Mello's discovery was particularly notable because it represented the first identification of the causative agent for the phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine administered by the Nobel Foundation, is awarded once a year for outstanding discoveries in the field of life science and medicine. It is one of five Nobel Prizes established in 1895 by Swedish chemist Alfred Nobel, the inventor of dynamite, in his will...
in 2006 for their work.
External links
- RNAi Atlas: a database of RNAi libraries and their target analysis results in Homo sapians
- Overview of the RNAi process, from Cambridge University's The Naked ScientistsThe Naked ScientistsThe Naked Scientists is a one-hour audience-interactive science radio talk show broadcast live by the BBC in the East of England, nationally by BBC Radio 5 Live and internationally as a podcast. The programme was created and is edited by Cambridge University Pathology Department clinical lecturer...
- Animation of the RNAi process, from NatureNature (journal)Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...
- NOVA scienceNOW explains RNAi – A 15 minute video of the NovaNOVA (TV series)Nova is a popular science television series from the U.S. produced by WGBH Boston. It can be seen on the Public Broadcasting Service in the United States, and in more than 100 other countries...
broadcast that aired on PBSPublic Broadcasting ServiceThe Public Broadcasting Service is an American non-profit public broadcasting television network with 354 member TV stations in the United States which hold collective ownership. Its headquarters is in Arlington, Virginia....
, July 26, 2005 - Silencing Genomes RNA interference (RNAi) experiments and bioinformatics in C. elegans for education. From the Dolan DNA Learning Center of Cold Spring Harbor Laboratory.
- RNAi screens in C. elegans in a 96-well liquid format and their application to the systematic identification of genetic interactions (a protocol)
- 2 American ‘Worm People’ Win Nobel for RNA Work, from NY TimesThe New York TimesThe New York Times is an American daily newspaper founded and continuously published in New York City since 1851. The New York Times has won 106 Pulitzer Prizes, the most of any news organization...
- Molecular Therapy web focus: "The development of RNAi as a therapeutic strategy" , a collection of free articles about RNAi as a therapeutic strategy.
- DesiRM: Designing of Complementary and Mismatch siRNAs for Silencing a Gene
- GenomeRNAi: a database of phenotypes from RNA interference screening experiments in Drosophila melanogaster and Homo sapians