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. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell. Genes are regulated at either the transcriptional or post-transcriptional level. Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.). Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNAi. Both transcriptional and post-transcriptional gene silencing are used to regulate endogenous genes. Mechanisms of gene silencing also protect the organism's genome from transposons and viruses. Gene silencing thus may be part of an ancient immune system protecting from such infectious DNA elements. Genes may be silenced by DNA methylation during meiosis, as in the filamentous fungus Neurospora crassa.

Specific studies of gene silencing There are several more terms related to specific topics of gene silencing: Transcriptional Gene Silencing: Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. It is an inheritance process independent of the classical Mendelian inheritance. Imprinted genes are either expressed only from the allele inherited from the mother (eg. H19 or CDKN1C), or in other instances from the allele inherited from the father (eg. IGF2). Forms of genomic imprinting have been demonstrated in insects, mammals and flowering plants.

Overview In diploid organisms somatic cells possess two copies of the genome. Each autosomal gene is therefore represented by two copies, or alleles, with one copy inherited from each parent at fertilisation. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. In mammals however, a small proportion (<1%) of genes are imprinted, meaning that gene expression occurs from only one allele.[1] The expressed allele is dependent upon its parental origin. For example, the gene encoding Insulin-like growth factor 2 (IGF2/Igf2) is only expressed from the allele inherited from the father.[2] The phrase "imprinting" was first used to describe events in the insect Pseudococcus nipae.[3] In Pseudococcids or mealybugs (Homoptera, Coccoidea) both the male and female develop from a fertilised egg. In females, all chromosomes remain euchromatic and functional. In embryos destined to become males, one haploid set of chromosomes becomes heterochromatinised after the sixth cleavage division and remains so in most tissues; males are thus functionally haploid.[4][5][6] In insects, imprinting describes the silencing of the paternal genome in males, and thus is involved in sex determination. In mammals, genomic

imprinting describes the processes involved in introducing functional inequality between two parental alleles of a gene.[7]

[edit] Imprinted genes in mammals That imprinting might be a feature of mammalian development was suggested in breeding experiments in mice carrying reciprocal translocations.[8] Nucleus transplantation experiments in mouse zygotes in the early 1980s confirmed that normal development requires the contribution of both the maternal and paternal genomes. The vast majority of mouse parthenogenones/gynogenones (with two maternal or egg genomes) and androgenones (with two paternal or sperm genomes) die at, or before, the blastocyst/implantation stage. In the rare instances that they develop to postimplantation stages, gynogenetic embryos show better embryonic development relative to placental development, while for androgenones, the reverse is true. Nevertheless, for the latter, only a few have been described.[9][10][11] Parthenogenetic/gynogenetic embryos have twice the normal expression level of maternally derived genes, and lack expression of paternally expressed genes, while the reverse is true for androgenetic embryos. It is now known that there are at least 80 imprinted genes in humans and mice, many of which are involved in embryonic and placental growth and development.[12][13][14][15] No naturally occurring cases of parthenogenesis exist in mammals because of imprinted genes. Experimental manipulation of a paternal methylation imprint controlling the Igf2 gene has, however, recently allowed the creation of rare individual mice with two maternal sets of chromosomes - but this is not a true parthenogenone. Hybrid offspring of two species may exhibit unusual growth due to the novel combination of imprinted genes.[16]

[edit] Genetic mapping of imprinted genes At the same time as the generation of the gynogenetic and androgenetic embryos discussed above, mouse embryos were also being generated that contained only small regions that were derived from either a paternal or maternal source.[17][18] The generation of a series of such uniparental disomies, which together span the entire genome, allowed the creation of an imprinting map.[19] Those regions which when inherited from a single parent result in a discernible phenotype contain imprinted gene(s). Further research showed that within these regions there were often numerous imprinted genes.[20] Around 80% of imprinted genes are found in clusters such as these, called imprinted domains, suggesting a level of co-ordinated control.[21]

[edit] Imprinting mechanisms This section does not cite any references or sources. Please help improve this article by adding citations to reliable sources. Unverifiable material may be challenged and removed. (July 2008) Imprinting is a dynamic process. It must be possible to erase and re-establish the imprint through each generation. The nature of the imprint must therefore be epigenetic (modifications to the structure of the DNA rather than the sequence). In germline cells the imprint is erased, and then re-established according to the sex of the individual; i.e. in the developing sperm, a paternal imprint is established, whereas in developing oocytes, a maternal imprint is established. This process of erasure and reprogramming is necessary such that the current imprinting status is relevant to the sex of the individual. In both plants and mammals there are two major mechanisms that are involved in establishing the imprint; these are DNA methylation and histone modifications.

[edit] Regulation

This section does not cite any references or sources. Please help improve this article by adding citations to reliable sources. Unverifiable material may be challenged and removed. (July 2008) The grouping of imprinted genes within clusters allows them to share common regulatory elements, such as non-coding RNAs and differentially methylated regions (DMRs). When these regulatory elements control the imprinting of one or more genes, they are known as imprinting control regions (ICR). The expression of non-coding RNAs, such as Air on mouse chromosome 17 and KCNQ1OT1 on human chromosome 11p15.5, have been shown to be essential for the imprinting of genes in their corresponding regions. Differentially methylated regions are generally segments of DNA rich in cytosine and guanine nucleotides, with the cytosine nucleotides methylated on one copy but not on the other. Contrary to expectation, methylation does not necessarily mean silencing; instead, the effect of methylation depends upon the default state of the region.

[edit] Functions of imprinted genes The control of expression of specific genes by genomic imprinting is unique to therian mammals (placental mammals and marsupials) and flowering plants. Imprinting of whole chromosomes has been reported in mealybugs.[3][4][5][6] and a fungus gnat (Sciara).[22] It has also been established that X-chromosome inactivation occurs in an imprinted manner in the extra-embryonic tissues of mice, where it is always the paternal X-chromosome which is silenced.[21][23] The majority of imprinted genes in mammals have been found to have roles in the control of embryonic growth and development, including development of the placenta.[12][24] Other imprinted genes are involved in post-natal development, with roles affecting suckling and metabolism.[24][25]

[edit] Theories on the origins of imprinting Imprinting appears to be able to increase the evolutionary fitness of genes in two ways, so either or both could be responsible for its origins. A widely accepted hypothesis for the occurrence of genomic imprinting is the "parental conflict hypothesis."[26] This hypothesis states that the inequality between parental genomes due to imprinting is a result of the differing interests of each parent in terms of the evolutionary fitness of their genes. The father is more 'interested' in the growth of his offspring, at the expense of the mother. The mother's interest is to conserve resources for her own survival while providing sufficient nourishment to current and subsequent litters. Accordingly, paternally expressed genes tend to be growth promoting whereas maternally expressed genes tend to be growth limiting.[26] Another hypothesis behind the origins of genomic imprinting is that this phenomenon evolved to silence foreign DNA elements, such as genes of viral origin. There appears to be an over-representation of retrotransposed genes, that is to say genes that are inserted into the genome by viruses, among imprinted genes. It has also been postulated that if the retrotransposed gene is inserted close to another imprinted gene, it may just acquire this imprint.[27]

[edit] Problems associated with imprinting Imprinting may cause problems in cloning, with clones having DNA that is not methylated in the correct position. It is possible that this is due to a lack of time for reprogramming to be completely achieved. When a nucleus is added to an egg during somatic cell nuclear transfer, the egg starts dividing in minutes, as compared to the days or months it takes for

reprogramming during embryonic development. If time is the responsible factor, it may be possible to delay cell division in clones, giving time for proper reprogramming to occur. An allele of the "callipyge" (from the Greek for "beautiful buttocks"), or CLPG, gene in sheep produces large buttocks consisting of muscle with very little fat. The large-buttocked phenotype only occurs when the allele is present on the copy of chromosome 18 inherited from a sheep's father and is not on the copy of chromosome 18 inherited from that sheep's mother.[28]

[edit] Examples Several genetic diseases that map to 15q11 (band 11 of the long arm of chromosome 15) in humans are due to abnormal imprinting. This region is differently imprinted in maternal and paternal chromosomes, and both imprintings are needed for normal development. In a normal individual, the maternal allele is methylated, while the paternal allele is unmethylated. It is possible for an individual to fail to inherit a properly imprinted 15q11 from one parent, as a result either of deletion of the 15q11 region from that parent's chromosome 15 or, less frequently, of uniparental disomy (in which both copies have been taken from the other parent's genes). •

If neither copy of 15q11 has paternal imprinting, the result is Prader-Willi syndrome (characterised by hypotonia, obesity, and hypogonadism).

•

If neither copy has maternal imprinting, the result is Angelman syndrome (characterised by epilepsy, tremors, and a perpetually smiling facial expression).

[edit] NOEY2 NOEY2 is a paternally expressed imprinted gene located on chromosome 1 in humans. Loss of NOEY2 expression is linked to an increased risk of ovarian and breast cancers; in 41% of breast and ovarian cancers the protein transcribed by NOEY2 is not expressed, suggesting that it functions as a tumor suppressor gene[29] Therefore, if a person inherits both chromosomes from the mother, the gene will not be expressed and the individual is put at a greater risk for breast and ovarian cancer.

[edit] Imprinted genes in plants Decades after imprinting was demonstrated in the mouse, a similar phenomenon was observed in flowering plants (angiosperms). During fertilisation of the embryo in flowers, a second separate fertilisation event gives rise to the endosperm, an extraembryonic structure that nourishes the seed similar to the mammalian placenta. Unlike the embryo, the endosperm often contains two copies of the maternal genome and fusion with a male gamete results in a triploid genome. This uneven ratio of maternal to paternal genomes appears to be critical for seed development. Some genes are found to be expressed from both maternal genomes while others are expressed exclusively from the lone paternal copy.[30]

Paramutation From Wikipedia, the free encyclopedia Jump to: navigation, search

In epigenetics, paramutation is an interaction between two alleles of a single locus, resulting in a heritable change of one allele that is induced by the other allele. Paramutation violates Mendel’s first law, which states that in the process of the formation of the gametes (egg or sperm) the allelic pairs separate, one going to each gamete, and that each gene remains

completely uninfluenced by the other. In paramutation an allele in one generation heritably affects the other allele in future generations, even if the allele causing the change is itself not transmitted. What may be transmitted in such a case are RNAs such as piRNAs, siRNAs, miRNAs or other regulatory RNAs. These are packaged in egg or sperm and cause paramutation upon transmission to the next generation. This means that RNA is a molecule of inheritance, just like DNA. Paramutation was first discovered and studied in maize (Zea mays) by R.A. Brink at the University of Wisconsin-Madison in the 1950s. Brink noticed that specific weakly expressed alleles of the red1 (r1) locus in maize, which confers red pigment to corn kernels, can heritably change specific strongly expressed alleles to a weaker expression state. The weaker expression state adopted by the changed allele is heritable and can, in turn, change the expression state of other active alleles in a process termed secondary paramutation. Brink showed that the influence of the paramutagenic allele could persist for many generations. However, since paramutation is an epigenetic phenomenon, it can be attenuated over successive generations by dilution of the causative RNA. This contrasts sharply with ordinary mutations in DNA, which do not fade away. Interestingly, paramutation can result in a single allele of a gene controlling a spectrum of phenotypes. At r1 in maize, for example, the weaker expression state adopted by an allele following paramutation can range from completely colorless to nearly fully-colored kernels. This is an exception to the rule that continuous variation is controlled by many genes (see multi-genic traits). Allelic interactions similar to paramutation have since been reported in other organisms, including tomato, pea, and mice. The molecular basis of paramutation is being unraveled. Paramutation may share common mechanisms with other epigenetic phenomena, such as gene silencing, genomic imprinting, and transvection (genetics). Alleman (2006) proposed that "paramutation is RNA-directed. Stability of the chromatin states associated with paramutation and transposon silencing requires the mop1 gene, which encodes an RNA-dependent RNA polymerase." This polymerase is required to maintain a threshold level of the repeat RNA, which causes the paramutation. Exactly how the RNA does this is not understood, but like other epigenetic changes, it involves a covalent modification of the DNA and/or the DNA-bound histone proteins without changing the DNA sequence of the gene itself. Post-transcriptional Gene Silencing: Post transcriptional gene silencing (PTGS) is a mechanism for sequence-specific RNA degradation in plants similar to RNA interference in other organisms. The process was described first in transgenic Petunia. The scientists’ goal was to produce petunia plants with improved flower colors. To achieve this goal, they introduced additional copies of a gene encoding a key enzyme for flower pigmentation into petunia plants. Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple or deep red flowers but carried fully white or partially white flowers. When the scientists had a closer look they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. Because of this observation the phenomenon was first named “co-suppression of gene expression” but the molecular mechanism remained unknown. A few years later plant virologists made a similar observation. In their research they aimed towards improvement of resistance of plants against plant viruses. At that time it was known that plants expressing virus-specific proteins show enhanced tolerance or even resistance against virus infection. However, they also made the surprising observation that plants carrying only short regions of viral RNA sequences not coding for any viral protein showed

the same effect. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from multiplying and spreading throughout the plant. They did the reverse experiment and put short pieces of plant gene sequences into plant viruses. Indeed, after infection of plants with these modified viruses the expression of the targeted plant gene was suppressed. They called this phenomenon “virus-induced gene silencing” or simply “VIGS”. RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are. Two types of small 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 specific other RNAs and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes – viruses and transposons – but also in directing development as well as gene expression in general. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA molecules into short fragments of ~20 nucleotides. One of the two strands of each fragment, known as the guide strand, is then 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. This process is known to spread systemically throughout the organism despite 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. Historically, RNA interference was known by other names, including post transcriptional gene silencing, 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,[2] which they published in 1998.[3]

Cellular mechanism

The dicer protein from Giardia intestinalis, which catalyzes the cleavage of dsRNA to siRNAs. The RNase domains are colored green, the PAZ domain yellow, the platform domain red, and the connector helix blue.[4] 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.[2] 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 (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 to be cleaved by dicer.

Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC complex.[5]

[edit] dsRNA cleavage Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer,[6] which binds and cleaves double-stranded RNAs (dsRNA)s to produce double-stranded fragments of 20– 25 base pairs[not in citation given] with a few unpaired overhang bases on each end.[7][8] Bioinformatics studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects.[9] 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.[10] 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.[11] This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown.[11] These RNA-binding proteins then facilitate transfer of cleaved siRNAs to the RISC complex.[12] 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.[13] These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).[14][15]

[edit] MicroRNA

The stem-loop secondary structure of a pre-microRNA from Brassica oleracea. MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development.[16][17] 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 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.[18] 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.[19] In

Drosophila and C. elegans, miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.[20][21]

Left: A full-length argonaute protein from the archaea species Pyrococcus furiosus. Right: The PIWI domain of an argonaute protein in complex with double-stranded RNA.

[edit] RISC activation and catalysis The active components of an RNA-induced silencing complex (RISC) are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA.[2] 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.[22] Although it was first believed that an ATP-dependent helicase separated these two strands,[23] the process is actually ATP-independent and performed directly by the protein components of RISC.[24][25] The strand selected as the guide tends to be the one whose 5' end is least paired to its complement,[26] but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation.[27] Instead, the R2D2 protein may serve as the differentiating factor by binding the more-stable 5' end of the passenger strand.[28] 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.[29] 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.[30] Indeed, RNAi may be more effective against mRNA targets that are not translated.[31] 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;[32] miRNA activity is also clustered in P-bodies.[33] Disruption of P-bodies decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.[34]

[edit] Transcriptional silencing Components of the RNA interference pathway are also used in many eukaryotes in the maintenance of the organisation and structure of their genomes. Modification of histones and associated induction of heterochromatin formation serves to downregulate genes pretranscriptionally;[35] 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.[36] As a consequence, the induction and spread of heterochromatic regions requires the argonaute and RdRP proteins.[37] Indeed, deletion of these genes in the fission yeast S. pombe disrupts histone methylation and centromere formation,[38] causing slow or stalled anaphase during cell division.[39] In some cases, similar processes associated with histone modification have been observed to transcriptionally upregulate genes.[40] 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.[41] 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.[42] 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.[43]

[edit] Crosstalk with RNA editing The type of RNA editing that is most prevalent in higher eukaryotes converts adenosine nucleotides into inosine in dsRNAs via the enzyme adenosine deaminase (ADAR).[44] It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate.[45] Indeed, some pre-miRNAs do undergo A→I RNA editing,[46][47] and this mechanism may regulate the processing and expression of mature miRNAs.[47] Furthermore, at least one mammalian ADAR can sequester siRNAs from RNAi pathway components.[48] 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.[49]

[edit] Variation among organisms

Illustration of the major differences between plant and animal gene silencing. Natively expressed microRNA or exogenous small interfering RNA is processed by dicer and integrated into the RISC complex, which mediates gene silencing.[50] 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 (channels in the cell walls that enable communication and transport).[23] The heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell.[51] 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.[50] This translational effect may be produced by inhibiting the interactions of translation initiation factors with the messenger RNA's polyadenine tail.[52] Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi lack the RNAi pathway entirely.[53][54] Most or all of the components are also missing in some fungi, most notably the model organism Saccharomyces cerevisiae.[55] 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 niches.[56]

[edit] 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.[57] It has been suggested that CRISPR systems in prokaryotes are analogous to eukaryotic RNA interference systems, although none of the protein components are orthologous.[58]

[edit] Biological functions [edit] 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.[59] 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.[60] 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.[61] 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.[62] In response, many plant viruses have evolved elaborate mechanisms that suppress the RNAi response in plant cells.[63] 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.[64] Some plant genomes also express endogenous siRNAs in response to infection by specific types of bacteria.[65] These effects may be part of a generalized response to pathogens that downregulates any metabolic processes in the host that aid the infection process.[66]

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.[67][68] 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.[69][70] 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 RNAidependent mammalian immune response.[71][72] However, this hypothesis of RNAi-mediated immunity in mammals has been challenged as poorly substantiated.[73] 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.[40]

[edit] Downregulation of genes Endogenously expressed miRNAs, including both intronic and intergenic miRNAs, are most important in translational repression[50] 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 cells.[74] The role of endogenously expressed miRNA in downregulating gene expression was first described in C. elegans in 1993.[75] 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.[76] In plants, the majority of genes regulated by miRNAs are transcription factors;[77] 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 proteins.[78] In many organisms, including humans, miRNAs have also been linked to the formation of tumors and dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and tumor suppressors.[79]

[edit] Upregulation of genes RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter can increase gene transcription, a phenomenon dubbed RNA activation. Part of the mechanism for how these RNA upregulate genes is known: dicer and argonaute are involved, and there is histone demethylation.[80][81]

[edit] 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.[82] 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.[83] 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.[82][84] 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.[82] 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.[63] 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.[85]

[edit] Technological applications

A normal adult Drosophila fly, a common model organism used in RNAi experiments.

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.[86]

[edit] Gene knockdown 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.[2] 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.[87] 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.[9] A multitude of software tools have been developed implementing algorithms for the design of general,[88][89] mammal-specific,[90] and virusspecific[91] 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.[92] 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.[93] 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,[94] or by more elaborate lentiviral vector systems allowing the inducible activation or deactivation of transcription, known as conditional RNAi.[95][96]

[edit] Functional genomics Most functional genomics applications of RNAi in animals have used C. elegans[97] and Drosophila,[98] 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.[99] 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.[100] 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[101] and to predict their likely efficiency at gene knockdown.[102] 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.[103][104] 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.[105][106] 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)[107] as well as more common plant model systems Arabidopsis and maize.[108]

[edit] Medicine 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.[109] Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus,[110] RNAi has also been shown to be effective in the reversal of induced liver failure in mouse models.[111]

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,[112] knockdown of host receptors and coreceptors for HIV,[113] the silencing of hepatitis A[114] and hepatitis B genes,[115] silencing of influenza gene expression,[40] and inhibition of measles viral replication.[116] Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease.[117] 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.[118][119] 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.[120][121] 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.[122] A computational genomics study estimated that the error rate of off-target interactions is about 10%.[9] 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,[123] 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.

[edit] Biotechnology RNA interference has been used for applications in biotechnology, 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.[124] Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.[125] Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants[126] and decreased the precursors of likely carcinogens in tobacco plants.[127] Other plant traits that have been engineered in the laboratory include the production of nonnarcotic natural products by the opium poppy,[128] resistance to common plant viruses,[129] and fortification of plants such as tomatoes with dietary antioxidants.[130] 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.[131][132]

[edit] History and discovery

Example petunia plants in which genes for pigmentation are silenced by RNAi. The left plant is wild-type; the right plants contain transgenes that induce suppression of both transgene and endogenous gene expression, giving rise to the unpigmented white areas of the flower.[133]

Craig Mello at the 2006 Nobel Prize lecture. The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants,[134] and more directly by reports of unexpected outcomes in experiments performed by plant scientists in the U.S. and The Netherlands in the early 1990s.[135] In an attempt to alter flower colors 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 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,[136] 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.[137] This phenomenon was called co-suppression of gene expression, but the molecular mechanism remained unknown.[138] 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 virusspecific 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.[139] 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.[140] After these initial observations in plants, many laboratories around the world searched for the occurrence of this phenomenon in other organisms.[141][142] Craig C. Mello and Andrew Fire's 1998 Nature paper reported a potent gene silencing effect after injecting double stranded RNA into C. elegans.[3] 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.[2] Meiotic gene silencing:

Transvection is an epigenetic phenomenon that results from an interaction between an allele on one chromosome and the corresponding allele on the homologous chromosome. Transvection can lead to either gene activation or repression. Formally (see quote from Lewis, below), it can also occur between nonallelic regions of the genome as well as regions of the genome that are not transcribed. Edward B. Lewis at Caltech discovered transvection at the bithorax complex in Drosophila in the 1950s. Since then, transvection has been observed at a number of additional loci in Drosophila, including white, decapentaplegic, eyes absent, vestigial, and yellow. As stated by Ed Lewis, "Operationally, transvection is occurring if the phenotype of a given genotype can be altered solely by disruption of somatic (or meiotic) pairing. Such disruption can generally be accomplished by introduction of a heterozygous rearrangement that disrupts pairing in the relevant region but has no position effect of its own on the phenotype" (cited in Wu and Morris 1999). Recently, pairing-mediated phenomena have been observed in species other than Drosophila, including mice, humans, plants, nematodes, insects, and fungi. In light of these findings, transvection may represent a potent and widespread form of gene regulation. Interestingly, transvection appears to be dependent upon chromosome pairing. In some cases, if one allele is placed on a different chromosome by a translocation, transvection does not occur. Transvection can sometimes be restored in a translocation homozygote, where both alleles may once again be able to pair. Restoration of phenotype has been observed at bithorax, decapentaplegic, eyes absent, and vestigial, and with transgenes of white. In some cases, transvection between two alleles leads to intragenic complementation while disruption of transvection disrupts the complementation. Transvection is believed to occur through a variety of mechanisms. In one mechanism, the enhancers of one allele activate the promoter of a paired second allele. Other mechanisms include pairing-sensitive silencing and enhancer bypass of a chromatin insulator through pairing-mediated changes in gene structure. Cellular components of gene silencing: In biology, histones are the chief protein components of chromatin. They act as spools around which DNA winds, and they play a role in gene regulation. Without histones, the unwound DNA in chromosomes would be very long. For example, each human cell has about 1.8 meters of DNA, but wound on the histones it has about 90 millimeters of chromatin, which, when duplicated and condensed during mitosis, result in about 120 micrometers of chromosomes.[1] Chromatin is the complex combination of DNA, RNA, and protein that makes up chromosomes. It is found inside the nuclei of eukaryotic cells, and within the nucleoid in prokaryotic cells. It is divided between heterochromatin (condensed) and euchromatin (extended) forms.[1] [2] The major components of chromatin are DNA and histone proteins, although many other chromosomal proteins have prominent roles too. The functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis, and to serve as a mechanism to control expression and DNA replication. Chromatin contains genetic material-instructions to direct cell functions. Changes in chromatin structure are affected by chemical modifications of histone proteins such as methylation (DNA and proteins) and acetylation (proteins), and by non-histone, DNAbinding proteins. Heterochromatin is a tightly packed form of DNA. Its major characteristic is that transcription is limited. As such, it is a means to control gene expression, through regulation of the transcription initiation. Chromatin is found in two varieties: euchromatin and heterochromatin. Heterochromatin is believed to serve several functions, from gene

regulation to the protection of the integrity of chromosomes; all of these roles can be attributed to the dense packing of DNA, which makes it less accessible to protein factors that bind DNA or its associated factors. For example, naked double-stranded DNA ends would usually be interpreted by the cell as damaged DNA, triggering cell cycle arrest and DNA repair . In genetics, microRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. They were first described in 1993 by Lee and colleagues in the Victor Ambros lab [1], yet the term microRNA was only introduced in 2001 in a set of three articles in Science.[2] Small interfering RNA (SIRNA), sometimes known as short interfering RNA or silencing RNA, is a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated. siRNAs were first discovered by David Baulcombe's group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".[1] Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues in a paper published in Nature.[2] This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development. Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells. dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-stranded RNA such as viral RNA or siRNA can trigger RNA interference in eukaryotes, as well as interferon response in vertebrates.

Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA) about 20-25 nucleotides long, usually with a two-base overhang on the 3' end. Dicer contains two RNase III domains and one PAZ domain; the distance between these two regions of the molecule is determined by the length and angle of the connector helix and determines the length of the siRNAs it produces.[1] Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC), whose catalytic component argonaute is an endonuclease capable of degrading messenger RNA (mRNA) whose sequence is complementary to that of the siRNA guide strand.[2] The human version of this gene is DICER1. Dicer and other miRNA processing enzymes may be important in cancer prognosis. [3] Function: Involved in cleaving double-stranded RNA in the RNA interference (RNAi) pathway. It produces 21 to 23 bp dsRNAs (siRNAs) which target the selective destruction of homologous RNAs.

Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons were also once called "jumping genes", and are examples of mobile genetic elements. They were discovered by Barbara McClintock early in her career[1], for which she was awarded a Nobel prize in 1983. There are a variety of mobile genetic elements, and they can be grouped based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, copy themselves by first being transcribed to RNA, then transcribed back to DNA by reverse transcriptase, and then being inserted at another position in the genome. Class II mobile genetic elements move directly from one position to another using a transposase to "cut and paste" them within the genome. Transposons make up a large fraction of genome sizes which is evident through the C-values of eukaryotic species. They are very useful to researchers as a means to alter DNA inside of a living organism.