It is a biological process where an induced RNA or an alien RNA inhibits the functioning of another gene already present in the organism be it of a virus, a pathogen or the organism itself by causing typical destruction of its specific mRNA or messenger RNA molecules.
Definition / Scope
RNAi stands for RNA interference.
It is a biological process where an induced RNA or an alien RNA inhibits the funtioning of another gene already present in the organism be it of a virus, a pathogen or the organism itself by causing typical destruction of its specific mRNA or messenger RNA molecules. Historically it is known with other names which are post transcriptional gene silencing, or gene silencing and many more.
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 nucleotide sequences – viruses and transposons. It also influences development.
The RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double-stranded fragments of ~20 nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), the passenger strand and the guide strand.
RNAi is a valuable research tool, both in cell culture and in living organisms, because synthetic dsRNA introduced into cells can selectively and robustly induce suppression of specific genes of interest. RNAi may be used for large-scale screens that systematically shut down each gene in the cell, which can help to identify the components necessary for a particular cellular process or an event such as cell division. The pathway is also used as a practical tool in biotechnology, medicine and insecticides
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 Dicer. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding genes 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. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC.
ds RNA or double stranded RNA initiates the RNAi gene by activating the ribonuclease RNA protein dicer enzyme. This ribonuclease binds and cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 20–25 base pairs with a 2-nucleotide overhang at the 3′ end.
For a brief understanding we should understand the structure of a double stranded DNA .only after we have sufficient knowledge to undertsand the polarity of a ds-DNA and its properties can we understand the proper mechanism related with the RNA silencing.
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 (siRNAs). 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 cleave it, thereby preventing it from being used as a translation template.
The enzyme dicer trims double stranded RNA, to form small interfering RNA or microRNA. These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation.
Components of the RNAi pathway are used in many eukaryotes in the maintenance of the organization and structure of their genomes. Modification of histones 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. Most studies have focused on the mating-type region in fission yeast, which may not be representative of activities in other genomic regions/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 mammals is not clear, as heterochromatin maintenance in mammalian cells may be independent of the components of the RNAi pathway.
The RNA interference pathway is often exploited in experimental biology to study the function of genes in cell culture and in vivo in model organisms. 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.
Such problems occur more frequently when the dsRNA contains repetitive sequences. It has been estimated from studying the genomes of humans, C. elegans and S. pombe that about 10% of possible siRNAs have substantial off-target effects. A multitude of software tools have been developed implementing algorithms 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 mammalian 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 oocytes and cells from early mouse embryos lack this reaction to exogenous dsRNA and are therefore a common model system for studying mammalian gene-knockdown effects. 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.
A normal adult Drosophila fly, a common model organism used in RNAi experiments.
Most functional genomics applications of RNAi in animals have used C. elegans and Drosophila, as these are the common model organisms in which RNAi is most effective. C. elegans is particularly useful for RNAi research for two reasons: firstly, the effects of 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 networks 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 microarrays. 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.
An adult C. elegans worm, grown under RNAi suppression of a nuclear hormone receptor involved in desaturase regulation. These worms have abnormal fatty acid metabolism but are viable and fertile.
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 has been more successful. Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus. RNAi has also been shown to be effective in reversing induced liver failure in mouse models.
Potential antiviral therapies include topical microbicide treatments that use RNAi to treat infection (at Harvard 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 to polyglutamine diseases such as Huntington’s disease.
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.
RNA interference is also a promising way to treat cancers 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.
Due to safety concerns with viral vectors, nonviral delivery methods, typically employing lipid-based or polymeric vectors, are also promising candidates. Computational modeling of nonviral siRNA delivery paired with in vitro and in vivo gene knockdown studies elucidated the temporal behavior of RNAi in these systems.
The model used an input bolus dose of siRNA and computationally and experimentally showed that knockdown duration was dependent mainly on the doubling time of the cells to which siRNA was delivered, while peak knockdown depended primarily on the delivered dose. Kinetic considerations of RNAi are imperative to safe and effective dosing schedules as nonviral methods of inducing RNAi continue to be developed.
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 reported that 23 out of 49 distinct RNAi treatment protocols resulted in death. Researchers hypothesized this alarmingly high rate 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. Such considerations are under active investigation, to reduce their impact in the potential therapeutic applications.
RNAi in vivo delivery to tissues still eludes science—especially to tissues deep within the body. RNAi delivery is only easily accessible to surface tissues such as the eye and respiratory tract. In these instances, siRNA has been used in direct contact with the tissue for transport. The resulting RNAi successfully focused on target genes.
When delivering siRNA to deep tissues, the siRNA must be protected from nucleases, but targeting specific areas becomes the main difficulty. This difficulty has been combatted with high dosage levels of siRNA to ensure the tissues have been reached, however in these cases hepatotoxicity was reported.
RNA interference has been used for applications in biotechnology and is nearing commercialization in others. RNAi has developed many novel crops such as nicotinefree tobacco, decaffeinated coffee, nutrient fortified and hypoallergenic crops. The genetically engineered Arctic apples are near close to receive US approval.
The apples were produced by RNAi suppression of PPO (polyphenol oxidase) gene making apple varieties that will not undergo browning after being sliced. PPO-silenced apples are unable to convert chlorogenic acid into quinone product.
There are several opportunities for the applications of RNAi in crop science for its improvement such as stress tolerance and enhanced nutritional level. RNAi will prove its potential for inhibition of photorespiration to enhance the productivity of C3 plants. This knockdown technology may be useful in inducing early flowering, delayed ripening, delayed senescence, breaking dormancy, stress-free plants, overcoming self-sterility, etc.
RNAi has been used to genetically engineer plants to produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. 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 itself important in preventing damage from plant pests. Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.
No plant products that use RNAi-based genetic engineering have yet exited the experimental stage. Development efforts have successfully reduced the levels of allergens in tomato plants and fortification of plants such as tomatoes with dietary antioxidants. Previous commercial products, including the Flavr Savr tomato and two cultivars of ringspot-resistant papaya, were originally developed using antisense technology but likely exploited the RNAi pathway.
Another effort decreased the precursors of likely carcinogens 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 and resistance to common plant viruses.
RNAi is under development as an insecticide, employing multiple approaches, including genetic engineering and topical application. Cells in the midgut of many larvae take up the molecules and help spread the signal throughout the insect’s body.
RNAi has varying effects in different species of Lepidoptera (butterflies and moths). Possibly because their saliva is better at breaking down RNA, the cotton bollworm, the beet armyworm and the Asiatic rice borer have so far not been proven susceptible to RNAi by feeding.
To develop resistance to RNAi, the western corn rootworm would have to change the genetic sequence of its Snf7 gene at multiple sites. Combining multiple strategies, such as engineering the protein Cry, derived from a bacterium called Bacillus thuringiensis (Bt), and RNAi in one plant delay the onset of resistance.
One unconfirmed 2012 paper detected small RNAs from food plants in the blood of mice and humans. The consequences of RNA insecticides in the human bloodstream have not been investigated. Biological barriers—including saliva and blood enzymes and stomach acids may break down any ingested RNA.
Critics charge that the human equivalent of the mouse diet in the study would be 33 kilograms of cooked rice a day. Two 2013 studies failed to detect RNAs in humans. Athletes consuming a diet of apples and bananas and monkeys consuming a fruit shake both appeared to be RNA-free.
Transgenic crops have been made to express small bits of RNA, carefully chosen to silence crucial genes in target pests. RNAs exist that affect only insects that have specific genetic sequences. In 2009 a study showed RNAs that could kill any one of four fruit fly species while not harming the other three.
In 2012 Syngenta bought Belgian RNAi firm Devgen for $522 million and Monsanto paid $29.2 million for the exclusive rights to intellectual property from Alnylam Pharmaceuticals. The International Potato Center in Lima, Peru is looking for genes to target in the sweet potato weevil, a beetle whose larvae ravage sweet potatoes globally.
Other researchers are trying to silence genes in ants, caterpillars and pollen beetles. Monsanto will likely be first to market, with a transgenic corn seed that expresses dsRNA based on gene Snf7 from the western corn rootworm, a beetle whose larvae annually cause one billion dollars in damage in the United States alone. A 2012 paper showed that silencing Snf7 stunts larval growth, killing them within days. In 2013 the same team showed that the RNA affects very few other species.
Alternatively dsRNA can be supplied without genetic engineering. One approach is to add them to irrigation water. The molecules are absorbed into the plants’ vascular system and poison insects feeding on them. Another approach involves spraying RNA like a conventional pesticide. This would allow faster adaptation to resistance. Such approaches would require low cost sources of RNAs that do not currently exist.
Genome-scale RNAi research relies on high-throughput screening (HTS) technology. RNAi HTS technology allows genome-wide loss-of-function screening and is broadly used in the identification of genes associated with specific 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 major advantage of genome-scale RNAi screening is its ability to simultaneously interrogate thousands of genes. With the ability to generate a large amount of data per experiment, genome-scale RNAi screening has led to an explosion data generation rates. Exploiting such large data sets is a fundamental challenge, requiring suitable statistics/bioinformatics methods. The basic process of cell-based RNAi screening includes the choice of an RNAi library, robust and stable cell types, transfection with RNAi agents, treatment/incubation, signal detection, analysis and identification of important genes or therapeutical targets.
RNA based therapeutics has garnered significant attention in the recent years due to its potential to treat variety of chronic diseases such as cancer, diabetes, AIDS, Tuberculosis and certain cardiovascular conditions.
Despite being in the clinical research phase, the RNA based therapeutics is been explored as a promising treatment option for the diseases which are difficult to treat. Development of this therapeutics is based on promising technologies such as RNA interference technology (RNAi), antisense technology and SMaRT technology.
RNAi technology and antisense technology together are gaining prominence in the research industry, as these technologies provide base sequence to develop RNA drugs. RNAi technology works by causing destruction of specific mRNA molecules; whereas, antisense technology works by synthesizing strand of RNA from known gene sequence.
These newly synthesized RNA strands then itself binds to mRNA and make mRNA inactive. Gene silencing potential of RNA based therapeutics is the primary driver for the growth of this market. The other drivers include target specificity and selectivity of RNAi therapeutics, more intense product focus versus platform technologies and virtual drug development models that enable companies to reduce the research cost.
However, critical issues in drug delivery, high cost of research, and high failure rates are some of the major hurdles for the companies working in this field. Despite the restraints, RNA based therapeutics has the potential to grow due to increasing interest exhibited by the pharmaceutical industries for the commercialization of these therapies.
Quark Pharmaceuticals, Inc. (USA), Alnylam Pharmaceuticals, Inc. (USA), Dicerna Pharmaceuticals, Inc. (USA), Tekmira Pharmaceuticals Corp. (Canada), Benitec Biopharma Limited (Australia), Genzyme Corporation (USA), ISIS pharmaceuticals Inc (USA), Silence Therapeutics PLC (UK) and Cenix BioScience GmbH (Germany) are some of the key players in this market. ISIS pharmaceuticals, Inc. (USA) has focused its research activities on antisense technology and developed antisense therapeutics, namely Mipomersen and Fomivirsen.
This therapeutics is commercially available in the market and marketed as Kynamro and Vitravene respectively. Quark, Alnylam and silence therapeutics are other major players of this market with strong research pipeline in RNA based therapeutics.