RNA Interference: A Close View (With Diagram)

This article provides a close view on the RNA interference.

Introduction:

Cells use dicer to trim double stranded RNA to form small interfering RNA or microRNA.

An exogenous dsRNA or endogenous pre-miRNA can be processed by dicer and incorporated into the RNA-induced silencing complex (RISC), which targets single-stranded messenger RNA mol­ecules and triggers translational repression; incorporation into the RNA-induced transcriptional silencing complex (RITS) induces genome maintenance activities such as histone methylation and chromatin reorganization.

RNA interference (also called “RNA-mediated interference”, abbreviated RNAi) is a mecha­nism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to have evolved as a form of innate immunity against viruses and also plays a major role in regulating development and genome maintenance.

The RNAi pathway is initiated by the enzyme dicer, which cleaves double-stranded RNA (dsRNA) to short double-stranded fragments of 20-25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and base-pairs with complementary sequences.

The well-studied outcome of this recognition event is a form of post-transcriptional gene silencing. This occurs when the guide strand base pairs with a messenger RNA (mRNA) molecule and induces degradation of the mRNA by argonaute, the catalytic component of the RISC complex.

The short RNA fragments are known as small interfer­ing RNA (siRNA), when they derive from exogenous sources and microRNA (miRNA), when they are produced from RNA-coding genes in the cell’s own genome. The RNAi pathway has been particularly well-studied in certain model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the flowering plant Arabidopsis thaliana.

The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms; 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.

Historically, RNA interference was known by other names, including post transcriptional gene silencing, transgene silencing, and quelling. Only after these apparently-unrelated processes were fully understood it became clear that they all described the RNAi phenomenon.

RNAi has also been confused with antisense suppression of gene expression, which does not act catalytically to degrade mRNA but instead involves single-stranded RNA fragments physically binding to mRNA and blocking translation. 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.

Cellular Mechanisms:

RNAi is an RNA-dependent gene silencing process that is mediated by the RNA-induced silencing complex (RISC) and is initiated by short double- stranded RNA molecules in the 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 dicer.

The initiating dsRNA can also be endogenous, as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to the characteristic stem-loop structure of pre-miRNA in the nucleus, and then exported to the cytoplasm to be cleaved by dicer. Thus the two pathways for exogenous and endogenous dsRNA converge at the RISC complex, which mediates gene silencing effects.

dsRNA Cleavage:

Exogenous dsRNA initiates RNAi by activating the ribonuclease protein dicer, which binds and cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 20-25 base pairs with a few unpaired overhang bases on each 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 (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 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. These RNA-binding proteins then facilitate transfer of cleaved siRNAs to the RISC complex.

This initiation pathway may be amplified by the cell through the synthesis of a population of ‘secondary’ siRNAs using the dicer-produced initiating or ‘primary’ siRNAs as templates. These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).

microRNA:

MicroRNAs (miRNAs) are genomically encoded non-coding RNAs 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 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 Pasha. 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.

RISC Activation and Catalysis:

The catalytically-active components of the RISC complex are endonucleases 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 that with a more stable 5′-end, 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 less- 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 such as magnesium and by aromatic stacking 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 in cells decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.

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. elegans, although not in Drosophila or mammals. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata.

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 factors 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. Certain ascomycetes and basidiomycetes are also missing RNA interference pathways; this observation 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 niches.

Biological Functions:

Immunity:

RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants where it may also prevent self-propagation by transposons. Plants such as Arabidopsis thaliana express multiple dicer homologues 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 innate 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 down regulates 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 up-regulated 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.

Genome Maintenance:

Components of the RNA interference pathway are used in many eukaryotes in the maintenance of the organisation and structure of their genomes. Modification of histones and associated induction of heterochromatin formation serve to down regulate 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 chromo domain protein Chp1, and a protein called Task 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 up-regulate 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 mammals is not clear, as heterochromatin maintenance in mammalian cells may be independent of the components of the RNAi pathway.

miRNAs and Gene Regulation:

Endogenously expressed miRNAs, including both intronic and intergenic miRNAs, are most im­portant in translational repression and in the regulation of development, especially the timing of morphogenesis and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cells.

The role of endogenously expressed miRNA in down regulating 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 factors; thus miRNA activity is particularly wide-ranging and regulated entire gene networks during development by modulating the expression of key regulatory genes, including transcription factors as well as F- box proteins. In many organisms, including humans, miRNAs have also been linked to the formation of tumours and dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and tumour suppressors.

Crosstalk with RNA Editing:

The type of RNA editing that is most prevalent in higher eukaryotes convert’s 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→1 RNA editing may coun­teract RNAi silencing of endogenous genes and transgenes.

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 systems in prokaryotes are analogous to eukaryotic RNA inter­ference systems; although none of the protein components are orthologous.

Evolution:

Based on parsimony-based phylogenetic analysis, the most recent common ancestor of all eukaryotes most likely already possessed an early RNA interference pathway; the absence of the pathway in certain eukaryotes is thought to be a derived characteristic. The 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 transposons 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.

Gene Knockdown:

A wild-type adult Caenorhabditis elegans nematode 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. 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 to 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 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 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.

Functional Genomics:

Most functional genomics applications of RNAi in animals have used C. elegans and D. melanogaster, 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 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 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 the hexaploid wheat Triticum aestivum, as well as more common plant model systems Arabidopsis thaliana and Zea mays.

History and Discovery:

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 USA and The Netherlands in the early 1990s.

In an attempt to alter flower colours in petunias, 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 colour. 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 down regulated 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 down regulation 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 labelled “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 of a previously inexplicable phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work.