RNA interference


RNA interference

RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons (significant for some forms of innate immune response), and also plays a role in regulating development and genome maintenance. Small interfering RNA strands (siRNA) are key to the RNAi process, and have complementary nucleotide sequences to the targeted RNA strand. Specific RNAi pathway proteins are guided by the siRNA to the targeted messenger RNA (mRNA), where they "cleave" the target, breaking it down into smaller portions that can no longer be translated into protein. A type of RNA transcribed from the genome itself, microRNA (miRNA), works in the same way.cite book | author = Morris KV (editor). | title = RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity | publisher = Caister Academic Press | year = 2008 | url=http://www.horizonpress.com/rnareg | id = [http://www.horizonpress.com/rnareg ISBN 978-1-904455-25-7 ] ]

The RNAi pathway is initiated by the enzyme dicer, which cleaves long, dsRNA molecules into short 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 pairs with complementary sequences. The most well-studied outcome of this recognition event is post-transcriptional gene silencing. This occurs when the guide strand specifically pairs with an mRNA molecule and induces cleavage by argonaute, the catalytic component of the RISC complex. Another outcome is epigenetic changes to a gene – histone modification and DNA methylation – affecting the degree the gene is transcribed.

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. 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 protein 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",cite web | title=Advanced Information: RNA interference | last=Daneholt | first=Bertil | work=The Nobel Prize in Physiology or Medicine 2006 | url=http://nobelprize.org/nobel_prizes/medicine/laureates/2006/adv.html | accessdate=2007-01-25] which they published in 1998.cite journal |author=Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C |title=Potent and specific genetic interference by double-stranded RNA in "Caenorhabditis elegans" |journal=Nature |volume=391 |issue=6669 |pages=806–11 |year=1998 |pmid=9486653 |doi=10.1038/35888]

Cellular mechanism

RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component argonaute. When the dsRNA is exogenous (coming from infection by a virus with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by the enzyme 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.cite journal
author=Bagasra O, Prilliman KR
title=RNA interference: the molecular immune system
journal=J. Mol. Histol.
volume=35
issue=6
pages=545–53
year=2004
pmid=15614608
doi=10.1007/s10735-004-2192-8
url=http://www.kluweronline.com/art.pdf?issn=1567-2379&volume=35&page=545
]

dsRNA cleavage

Exogenous dsRNA initiates RNAi by activating the ribonuclease protein dicer,cite journal |author=Bernstein E, Caudy A, Hammond S, Hannon G |title=Role for a bidentate ribonuclease in the initiation step of RNA interference |journal=Nature |volume=409 |issue=6818 |pages=363–6 |year=2001 |pmid=11201747 |doi=10.1038/35053110] which binds and cleaves double-stranded RNAs (dsRNA)s to produce double-stranded fragments of 20–25 base pairs with a few unpaired overhang bases on each end.cite journal |author=Zamore P, Tuschl T, Sharp P, Bartel D |title=RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals |journal=Cell |volume=101 |issue=1 |pages=25–33 |year=2000 |pmid=10778853 |doi=10.1016/S0092-8674(00)80620-0] cite journal |author=Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall W, Karpilow J, Khvorova A |title=The contributions of dsRNA structure to dicer specificity and efficiency |journal=RNA |volume=11 |issue=5 |pages=674–82 |year=2005 |pmid=15811921 |doi=10.1261/rna.7272305] Bioinformatics studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects.cite journal |author=Qiu S, Adema C, Lane T |title=A computational study of off-target effects of RNA interference |journal=Nucleic Acids Res |volume=33 |issue=6 |pages=1834–47 |year=2005 |pmid=15800213 |doi=10.1093/nar/gki324] 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. [cite journal |author=Ahlquist P |title=RNA-dependent RNA polymerases, viruses, and RNA silencing |journal=Science |volume=296 |issue=5571 |pages=1270–3 |year=2002 |pmid=12016304 |doi=10.1126/science.1069132]

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.cite journal |author=Parker G, Eckert D, Bass B |title=RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA |journal=RNA |volume=12 |issue=5 |pages=807–18 |year=2006 |pmid=16603715 |doi=10.1261/rna.2338706] 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.cite journal |author=Liu Q, Rand T, Kalidas S, Du F, Kim H, Smith D, Wang X |title=R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway |journal=Science |volume=301 |issue=5641 |pages=1921–5 |year=2003 |pmid=14512631 |doi=10.1126/science.1088710]

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. [cite journal |author=Baulcombe D |title=Molecular biology. Amplified silencing |journal=Science |volume=315 |issue=5809 |pages=199–200 |year=2007 |pmid=17218517 |doi=10.1126/science.1138030] These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).cite journal |author=Pak J, Fire A |title=Distinct populations of primary and secondary effectors during RNAi in C. elegans |journal=Science |volume=315 |issue=5809 |pages=241–4 |year=2007 |pmid=17124291 |doi=10.1126/science.1132839] cite journal |author=Sijen T, Steiner F, Thijssen K, Plasterk R |title=Secondary siRNAs result from unprimed RNA synthesis and form a distinct class |journal=Science |volume=315 |issue=5809 |pages=244–7 |year=2007 |pmid=17158288 |doi=10.1126/science.1136699]

MicroRNA

MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development. [cite journal |author=Wang QL, Li ZH |title=The functions of microRNAs in plants |journal=Front. Biosci. |volume=12 |issue= |pages=3975–82 |year=2007 |pmid=17485351] [cite journal |author=Zhao Y, Srivastava D |title=A developmental view of microRNA function |journal=Trends Biochem. Sci. |volume=32 |issue=4 |pages=189–97 |year=2007 |pmid=17350266 |doi=10.1016/j.tibs.2007.02.006] 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.cite journal |author=Gregory R, Chendrimada T, Shiekhattar R |title=MicroRNA biogenesis: isolation and characterization of the microprocessor complex |journal=Methods Mol Biol |volume=342 |issue= |pages=33–47 |year=2006 |pmid=16957365]

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.cite journal | author = Pillai RS, Bhattacharyya SN, Filipowicz W | title = Repression of protein synthesis by miRNAs: how many mechanisms? | journal = Trends Cell Biol | volume = | issue = | pages = | year = | pmid = 17197185] In "Drosophila" and "C. elegans", miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.cite journal |author=Okamura K, Ishizuka A, Siomi H, Siomi M |title=Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways |journal=Genes Dev |volume=18 |issue=14 |pages=1655–66 |year=2004 |pmid=15231716 |doi=10.1101/gad.1210204] cite journal |author=Lee Y, Nakahara K, Pham J, Kim K, He Z, Sontheimer E, Carthew R |title=Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways |journal=Cell |volume=117 |issue=1 |pages=69–81 |year=2004 |pmid=15066283 |doi=10.1016/S0092-8674(04)00261-2]

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. 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.cite journal |author=Gregory R, Chendrimada T, Cooch N, Shiekhattar R |title=Human RISC couples microRNA biogenesis and posttranscriptional gene silencing |journal=Cell |volume=123 |issue=4 |pages=631–40 |year=2005 |pmid=16271387 |doi=10.1016/j.cell.2005.10.022] Although it was first believed that an ATP-dependent helicase separated these two strands,cite book | authors = Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J | title = Molecular Cell Biology | edition = 5th ed. | publisher = WH Freeman: New York, NY | year = 2004 | url = http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.TOC | id = ISBN 978-0716743668] the process is actually ATP-independent and performed directly by the protein components of RISC.cite journal |author=Matranga C, Tomari Y, Shin C, Bartel D, Zamore P |title=Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes |journal=Cell |volume=123 |issue=4 |pages=607–20 |year=2005 |pmid=16271386 |doi=10.1016/j.cell.2005.08.044] cite journal |author=Leuschner P, Ameres S, Kueng S, Martinez J |title=Cleavage of the siRNA passenger strand during RISC assembly in human cells |journal=EMBO Rep |volume=7 |issue=3 |pages=314–20 |year=2006 |pmid=16439995 |doi=10.1038/sj.embor.7400637] The strand selected as the guide tends to be the one whose 5' end is least paired to its complement, [cite journal | title=Asymmetry in the assembly of the RNAi enzyme complex| author=Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N, Zamore PD| journal=Cell| year=2003| volume=115| issue=2| pages=199–208| pmid=14567917 | doi=10.1016/S0092-8674(03)00759-1] but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation.cite journal |author=Preall J, He Z, Gorra J, Sontheimer E |title=Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila |journal=Curr Biol |volume=16 |issue=5 |pages=530–5 |year=2006 |pmid=16527750 |doi=10.1016/j.cub.2006.01.061] Instead, the R2D2 protein may serve as the differentiating factor by binding the more-stable 5' end of the passenger strand.cite journal |author=Tomari Y, Matranga C, Haley B, Martinez N, Zamore P |title=A protein sensor for siRNA asymmetry |journal=Science |volume=306 |issue=5700 |pages=1377–80 |year=2004 |pmid=15550672 |doi=10.1126/science.1102755]

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.cite journal |author=Ma J, Yuan Y, Meister G, Pei Y, Tuschl T, Patel D |title=Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein |journal=Nature |volume=434 |issue=7033 |pages=666–70 |year=2005 |pmid=15800629 |doi=10.1038/nature03514]

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.cite journal |author=Sen G, Wehrman T, Blau H |title=mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage |journal=Differentiation |volume=73 |issue=6 |pages=287–93 |year=2005 |pmid=16138829 |doi=10.1111/j.1432-0436.2005.00029.x] Indeed, RNAi may be more effective against mRNA targets that are not translated.cite journal |author=Gu S, Rossi J |title=Uncoupling of RNAi from active translation in mammalian cells |journal=RNA |volume=11 |issue=1 |pages=38–44 |year=2005 |pmid=15574516 |doi=10.1261/rna.7158605] 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;cite journal |author=Sen G, Blau H |title=Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies |journal=Nat Cell Biol |volume=7 |issue=6 |pages=633–6 |year=2005 |pmid=15908945 |doi=10.1038/ncb1265] miRNA activity is also clustered in P-bodies.cite journal |author=Lian S, Jakymiw A, Eystathioy T, Hamel J, Fritzler M, Chan E |title=GW bodies, microRNAs and the cell cycle |journal=Cell Cycle |volume=5 |issue=3 |pages=242–5 |year=2006 |pmid=16418578] Disruption of P-bodies decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.cite journal |author=Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, Hamel J, Fritzler M, Chan E |title=Disruption of P bodies impairs mammalian RNA interference |journal=Nat Cell Biol |volume=7 |issue=12 |pages=1267–74 |year=2005 |pmid=16284622 |doi=10.1038/ncb1334]

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 pre-transcriptionally;cite journal |author=Holmquist G, Ashley T |title=Chromosome organization and chromatin modification: influence on genome function and evolution |journal=Cytogenet Genome Res |volume=114 |issue=2 |pages=96–125 |year=2006 |pmid=16825762 |doi=10.1159/000093326] 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.cite journal |author=Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal S, Moazed D |title=RNAi-mediated targeting of heterochromatin by the RITS complex |journal=Science |volume=303 |issue=5658 |pages=672–6 |year=2004 |pmid=14704433 |doi=10.1126/science.1093686] As a consequence, the induction and spread of heterochromatic regions requires the argonaute and RdRP proteins.cite journal |author=Irvine D, Zaratiegui M, Tolia N, Goto D, Chitwood D, Vaughn M, Joshua-Tor L, Martienssen R |title=Argonaute slicing is required for heterochromatic silencing and spreading |journal=Science |volume=313 |issue=5790 |pages=1134–7 |year=2006 |pmid=16931764 |doi=10.1126/science.1128813] Indeed, deletion of these genes in the fission yeast "S. pombe" disrupts histone methylation and centromere formation,cite journal |author=Volpe T, Kidner C, Hall I, Teng G, Grewal S, Martienssen R |title=Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi |journal=Science |volume=297 |issue=5588 |pages=1833–7 |year=2002 |pmid=12193640 |doi=10.1126/science.1074973] causing slow or stalled anaphase during cell division.cite journal |author=Volpe T, Schramke V, Hamilton G, White S, Teng G, Martienssen R, Allshire R |title=RNA interference is required for normal centromere function in fission yeast |journal=Chromosome Res |volume=11 |issue=2 |pages=137–46 |year=2003 |pmid=12733640 |doi=10.1023/A:1022815931524] In some cases, similar processes associated with histone modification have been observed to transcriptionally upregulate genes.Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R. (2006). Small dsRNAs induce transcriptional activation in human cells. "Proc Natl Acad Sci USA" 103(46):17337–42. PMID 17085592]

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.cite journal |author=Noma K, Sugiyama T, Cam H, Verdel A, Zofall M, Jia S, Moazed D, Grewal S |title=RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing |journal=Nat Genet |volume=36 |issue=11 |pages=1174–80 |year=2004 |pmid=15475954 |doi=10.1038/ng1452] 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.cite journal |author=Sugiyama T, Cam H, Verdel A, Moazed D, Grewal S |title=RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production |journal=Proc Natl Acad Sci USA |volume=102 |issue=1 |pages=152–7 |year=2005 |pmid=15615848 |doi=10.1073/pnas.0407641102] 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.cite journal |author=Wang F, Koyama N, Nishida H, Haraguchi T, Reith W, Tsukamoto T |title=The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells |journal=Mol Cell Biol |volume=26 |issue=11 |pages=4028–40 |year=2006 |pmid=16705157 |doi=10.1128/MCB.02189-05]

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).cite journal |author=Bass B |title=RNA editing by adenosine deaminases that act on RNA |journal=Annu Rev Biochem |volume=71 |issue= |pages=817–46 |year=2002 |pmid=12045112 |doi=10.1146/annurev.biochem.71.110601.135501] It was originally proposed in 2000 that the RNAi and A→I RNA editing pathways might compete for a common dsRNA substrate.cite journal |author=Bass B |title=Double-stranded RNA as a template for gene silencing |journal=Cell |volume=101 |issue=3 |pages=235–8 |year=2000 |pmid=10847677 |doi=10.1016/S0092-8674(02)71133-1] Indeed, some pre-miRNAs do undergo A→I RNA editing,cite journal |author=Luciano D, Mirsky H, Vendetti N, Maas S |title=RNA editing of a miRNA precursor |journal=RNA |volume=10 |issue=8 |pages=1174–7 |year=2004 |pmid=15272117 |doi=10.1261/rna.7350304] cite journal |author=Yang W, Chendrimada T, Wang Q, Higuchi M, Seeburg P, Shiekhattar R, Nishikura K |title=Modulation of microRNA processing and expression through RNA editing by ADAR deaminases |journal=Nat Struct Mol Biol |volume=13 |issue=1 |pages=13–21 |year=2006 |pmid=16369484 |doi=10.1038/nsmb1041] 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.cite journal |author=Yang W, Wang Q, Howell K, Lee J, Cho D, Murray J, Nishikura K |title=ADAR1 RNA deaminase limits short interfering RNA efficacy in mammalian cells |journal=J Biol Chem |volume=280 |issue=5 |pages=3946–53 |year=2005 |pmid=15556947 |doi=10.1074/jbc.M407876200] 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.cite journal |author=Nishikura K |title=Editor meets silencer: crosstalk between RNA editing and RNA interference |journal=Nat Rev Mol Cell Biol |volume=7 |issue=12 |pages=919–31 |year=2006 |pmid=17139332 |doi=10.1038/nrm2061]

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 (channels in the cell walls that enable communication and transport). The heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell. [cite journal | author=Jones L, Ratcliff F, Baulcombe DC| title=RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance| journal=Current Biology| year=2001| volume=11| issue=10| pages=747–757| url=http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRT-433PCG6-K&_user=10&_coverDate=05%2F15%2F2001&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1e8ad684cc54f07e94776926599d292b | doi=10.1016/S0960-9822(01)00226-3] 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.cite journal | author = Humphreys DT, Westman BJ, Martin DI, Preiss T | year = 2005 | title= MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. | journal = Proc Natl Acad Sci USA |volume=102 | pages=16961–16966 | pmid = 16287976 | doi = 10.1073/pnas.0506482102]

Some eukaryotic protozoa such as "Leishmania major" and "Trypanosoma cruzi" lack the RNAi pathway entirely.cite journal | author = DaRocha W, Otsu K, Teixeira S, Donelson J | title = Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi | journal = Mol Biochem Parasitol | volume = 133 | issue = 2 | pages = 175–86 | year = 2004 | pmid = 14698430 | doi = 10.1016/j.molbiopara.2003.10.005] cite journal | author = Robinson K, Beverley S | title = Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania | journal = Mol Biochem Parasitol | volume = 128 | issue = 2 | pages = 217–28 | year = 2003 | pmid = 12742588 | doi = 10.1016/S0166-6851(03)00079-3] Most or all of the components are also missing in some fungi, most notably the model organism "Saccharomyces cerevisiae".cite journal |author=L. Aravind, Hidemi Watanabe, David J. Lipman, and Eugene V. Koonin|title= Lineage-specific loss and divergence of functionally linked genes in eukaryotes |journal=Proceedings of the National Academy of Sciences|volume=97 |issue=21 |pages=11319–11324 |year=2000 |id= |doi=10.1073/pnas.200346997|pmid= 11016957] 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.cite journal |author=Nakayashiki H, Kadotani N, Mayama S |title=Evolution and diversification of RNA silencing proteins in fungi |journal=J Mol Evol |volume=63 |issue=1 |pages=127–35 |year=2006 |pmid=16786437 |doi=10.1007/s00239-005-0257-2]

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.cite journal |author=Morita T, Mochizuki Y, Aiba H |title=Translational repression is sufficient for gene silencing by bacterial small noncoding RNAs in the absence of mRNA destruction |journal=Proc Natl Acad Sci USA |volume=103 |issue=13 |pages=4858–63 |year=2006 |pmid=16549791 |doi=10.1073/pnas.0509638103] It has been suggested that CRISPR systems in prokaryotes are analogous to eukaryotic RNA interference systems, although none of the protein components are orthologous.cite journal |author=Makarova K, Grishin N, Shabalina S, Wolf Y, Koonin E |title=A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action |journal=Biol Direct |volume=1 |issue= |pages=7 |year=2006 |pmid=16545108 |doi=10.1186/1745-6150-1-7]

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.cite journal |author=Stram Y, Kuzntzova L |title=Inhibition of viruses by RNA interference |journal=Virus Genes |volume=32 |issue=3 |pages=299–306 |year=2006 |pmid=16732482 |doi=10.1007/s11262-005-6914-0] 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.cite journal |author=Blevins T, Rajeswaran R, Shivaprasad P, Beknazariants D, Si-Ammour A, Park H, Vazquez F, Robertson D, Meins F, Hohn T, Pooggin M |title=Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing |journal=Nucleic Acids Res |volume=34 |issue=21 |pages=6233–46 |year=2006 |pmid=17090584 |doi=10.1093/nar/gkl886] 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.cite journal |author=Palauqui J, Elmayan T, Pollien J, Vaucheret H |title=Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions |journal=EMBO J |volume=16 |issue=15 |pages=4738–45 |year=1997 |pmid=9303318 |doi=10.1093/emboj/16.15.4738] 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.cite journal |author=Voinnet O |title=RNA silencing as a plant immune system against viruses |journal=Trends Genet |volume=17 |issue=8 |pages=449–59 |year=2001 |pmid=11485817 |doi=10.1016/S0168-9525(01)02367-8] In response, many plant viruses have evolved elaborate mechanisms that suppress the RNAi response in plant cells.cite journal |author=Lucy A, Guo H, Li W, Ding S |title=Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus |journal=EMBO J |volume=19 |issue=7 |pages=1672–80 |year=2000 |pmid=10747034 |doi=10.1093/emboj/19.7.1672] 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.cite journal |author=Mérai Z, Kerényi Z, Kertész S, Magna M, Lakatos L, Silhavy D |title=Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing |journal=J Virol |volume=80 |issue=12 |pages=5747–56 |year=2006 |pmid=16731914 |doi=10.1128/JVI.01963-05] Some plant genomes also express endogenous siRNAs in response to infection by specific types of bacteria.cite journal |author=Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A, Zhu J, Staskawicz B, Jin H |title=A pathogen-inducible endogenous siRNA in plant immunity |journal=Proc Natl Acad Sci USA |volume=103 |issue=47 |pages=18002–7 |year=2006 |pmid=17071740 |doi=10.1073/pnas.0608258103] These effects may be part of a generalized response to pathogens that downregulates any metabolic processes in the host that aid the infection process.cite journal |author=Fritz J, Girardin S, Philpott D |title=Innate immune defense through RNA interference |journal=Sci STKE |volume=2006 |issue=339 |pages=pe27 |year=2006 |pmid=16772641 |doi=10.1126/stke.3392006pe27]

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.cite journal |author=Zambon R, Vakharia V, Wu L |title=RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster |journal=Cell Microbiol |volume=8 |issue=5 |pages=880–9 |year=2006 |pmid=16611236 |doi=10.1111/j.1462-5822.2006.00688.x] cite journal |author=Wang X, Aliyari R, Li W, Li H, Kim K, Carthew R, Atkinson P, Ding S |title=RNA interference directs innate immunity against viruses in adult Drosophila |journal=Science |volume=312 |issue=5772 |pages=452–4 |year=2006 |pmid=16556799 |doi=10.1126/science.1125694] 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.cite journal |author=Lu R, Maduro M, Li F, Li H, Broitman-Maduro G, Li W, Ding S |title=Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans |journal=Nature |volume=436 |issue=7053 |pages=1040–3 |year=2005 |pmid=16107851 |doi=10.1038/nature03870] cite journal |author=Wilkins C, Dishongh R, Moore S, Whitt M, Chow M, Machaca K |title=RNA interference is an antiviral defence mechanism in Caenorhabditis elegans |journal=Nature |volume=436 |issue=7053 |pages=1044–7 |year=2005 |pmid=16107852 |doi=10.1038/nature03957]

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.cite journal |author=Berkhout B, Haasnoot J |title=The interplay between virus infection and the cellular RNA interference machinery |journal=FEBS Lett |volume=580 |issue=12 |pages=2896–902 |year=2006 |pmid=16563388 |doi=10.1016/j.febslet.2006.02.070] cite journal |author=Schütz S, Sarnow P |title=Interaction of viruses with the mammalian RNA interference pathway |journal=Virology |volume=344 |issue=1 |pages=151–7 |year=2006 |pmid=16364746 |doi=10.1016/j.virol.2005.09.034] However, this hypothesis of RNAi-mediated immunity in mammals has been challenged as poorly substantiated.cite journal |author=Cullen B |title=Is RNA interference involved in intrinsic antiviral immunity in mammals? |journal=Nat Immunol |volume=7 |issue=6 |pages=563–7 |year=2006 |pmid=16715068 |doi=10.1038/ni1352] 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.cite journal |author=Li H, Ding S |title=Antiviral silencing in animals |journal=FEBS Lett |volume=579 |issue=26 |pages=5965–73 |year=2005 |pmid=16154568 |doi=10.1016/j.febslet.2005.08.034]

Downregulation of genes

Endogenously expressed miRNAs, including both intronic and intergenic miRNAs, are most important in translational repression and in the regulation of development, especially on the timing of morphogenesis and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cells.cite journal |author=Carrington J, Ambros V |title=Role of microRNAs in plant and animal development |journal=Science |volume=301 |issue=5631 |pages=336–8 |year=2003 |pmid=12869753 |doi=10.1126/science.1085242] The role of endogenously expressed miRNA in downregulating gene expression was first described in "C. elegans" in 1993.cite journal |author=Lee R, Feinbaum R, Ambros V |title=The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 |journal=Cell |volume=75 |issue=5 |pages=843–54 |year=1993 |pmid=8252621 |doi=10.1016/0092-8674(93)90529-Y] 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.cite journal |author=Palatnik J, Allen E, Wu X, Schommer C, Schwab R, Carrington J, Weigel D |title=Control of leaf morphogenesis by microRNAs |journal=Nature |volume=425 |issue=6955 |pages=257–63 |year=2003 |pmid=12931144 |doi=10.1038/nature01958] In plants, the majority of genes regulated by miRNAs are transcription factors;cite journal |author=Zhang B, Pan X, Cobb G, Anderson T |title=Plant microRNA: a small regulatory molecule with big impact |journal=Dev Biol |volume=289 |issue=1 |pages=3–16 |year=2006 |pmid=16325172 |doi=10.1016/j.ydbio.2005.10.036] 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.cite journal |author=Jones-Rhoades M, Bartel D, Bartel B |title=MicroRNAS and their regulatory roles in plants |journal=Annu Rev Plant Biol |volume=57 |issue= |pages=19–53 |year=2006 |pmid=16669754 |doi=10.1146/annurev.arplant.57.032905.105218] 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.cite journal |author=Zhang B, Pan X, Cobb G, Anderson T |title=microRNAs as oncogenes and tumor suppressors |journal=Dev Biol |volume=302 |issue=1 |pages=1–12 |year=2007 |pmid=16989803 |doi=10.1016/j.ydbio.2006.08.028]

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. [cite journal | author=Check E| title=RNA interference: hitting the on switch| journal=Nature| year=2007| volume=448| issue=7156| pages=855–858| pmid=17713502 | doi=10.1038/448855a] [cite journal |author=Li LC, Okino ST, Zhao H, "et al" |title=Small dsRNAs induce transcriptional activation in human cells |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=103 |issue=46 |pages=17337–42 |year=2006 |pmid=17085592 |url=http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17085592 |doi=10.1073/pnas.0607015103]

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.cite journal |author=Cerutti H, Casas-Mollano J |title=On the origin and functions of RNA-mediated silencing: from protists to man |journal=Curr Genet |volume=50 |issue=2 |pages=81–99 |year=2006 |pmid=16691418 |doi=10.1007/s00294-006-0078-x] 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.cite journal |author=Anantharaman V, Koonin E, Aravind L |title=Comparative genomics and evolution of proteins involved in RNA metabolism |journal=Nucleic Acids Res |volume=30 |issue=7 |pages=1427–64 |year=2002 |pmid=11917006 |doi=10.1093/nar/30.7.1427] 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.cite journal |author=Buchon N, Vaury C |title=RNAi: a defensive RNA-silencing against viruses and transposable elements |journal=Heredity |volume=96 |issue=2 |pages=195–202 |year=2006 |pmid=16369574 |doi=10.1038/sj.hdy.6800789] 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.cite journal |author=Obbard D, Jiggins F, Halligan D, Little T |title=Natural selection drives extremely rapid evolution in antiviral RNAi genes |journal=Curr Biol |volume=16 |issue=6 |pages=580–5 |year=2006 |pmid=16546082 |doi=10.1016/j.cub.2006.01.065]

Technological applications

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. 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.cite journal
author=Voorhoeve PM, Agami R
title=Knockdown stands up
journal=Trends Biotechnol.
volume=21
issue=1
pages=2–4
year=2003
pmid=12480342
url=http://linkinghub.elsevier.com/retrieve/pii/S0167779902000021
doi=10.1016/S0167-7799(02)00002-1
]

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,cite journal |author=Naito Y, Yamada T, Matsumiya T, Ui-Tei K, Saigo K, Morishita S |title=dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference |journal=Nucleic Acids Res |volume=33 |issue=Web Server issue |pages=W589–91 |year=2005 |pmid=15980542 |doi=10.1093/nar/gki419] cite journal |author=Henschel A, Buchholz F, Habermann B |title=DEQOR: a web-based tool for the design and quality control of siRNAs |journal=Nucleic Acids Res |volume=32 |issue=Web Server issue |pages=W113–20 |year=2004 |pmid=15215362 |doi=10.1093/nar/gkh408] mammal-specific,cite journal |author=Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K |title=siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference |journal=Nucleic Acids Res |volume=32 |issue=Web Server issue |pages=W124–9 |year=2004 |pmid=15215364 |doi=10.1093/nar/gkh442] and virus-specificcite journal |author=Naito Y, Ui-Tei K, Nishikawa T, Takebe Y, Saigo K |title=siVirus: web-based antiviral siRNA design software for highly divergent viral sequences |journal=Nucleic Acids Res |volume=34 |issue=Web Server issue |pages=W448–50 |year=2006 |pmid=16845046 |doi=10.1093/nar/gkl214] 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.cite journal |author=Reynolds A, Anderson E, Vermeulen A, Fedorov Y, Robinson K, Leake D, Karpilow J, Marshall W, Khvorova A |title=Induction of the interferon response by siRNA is cell type- and duplex length-dependent |journal=RNA |volume=12 |issue=6 |pages=988–93 |year=2006 |pmid=16611941 |doi=10.1261/rna.2340906] 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.cite journal |author=Stein P, Zeng F, Pan H, Schultz R |title=Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes |journal=Dev Biol |volume=286 |issue=2 |pages=464–71 |year=2005 |pmid=16154556 |doi=10.1016/j.ydbio.2005.08.015] 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,cite journal |author=Brummelkamp T, Bernards R, Agami R |title=A system for stable expression of short interfering RNAs in mammalian cells |journal=Science |volume=296 |issue=5567 |pages=550–3 |year=2002 |pmid=11910072 |doi=10.1126/science.1068999] or by more elaborate lentiviral vector systems allowing the inducible activation or deactivation of transcription, known as "conditional RNAi".cite journal |author=Tiscornia G, Tergaonkar V, Galimi F, Verma I |title=CRE recombinase-inducible RNA interference mediated by lentiviral vectors |journal=Proc Natl Acad Sci USA |volume=101 |issue=19 |pages=7347–51 |year=2004 |pmid=15123829 |doi=10.1073/pnas.0402107101] cite journal |author=Ventura A, Meissner A, Dillon C, McManus M, Sharp P, Van Parijs L, Jaenisch R, Jacks T |title=Cre-lox-regulated conditional RNA interference from transgenes |journal=Proc Natl Acad Sci USA |volume=101 |issue=28 |pages=10380–5 |year=2004 |pmid=15240889 |doi=10.1073/pnas.0403954101]

Functional genomics

Most functional genomics applications of RNAi in animals have used "C. elegans"cite journal |author=Kamath R, Ahringer J |title=Genome-wide RNAi screening in Caenorhabditis elegans |journal=Methods |volume=30 |issue=4 |pages=313–21 |year=2003 |pmid=12828945 |doi=10.1016/S1046-2023(03)00050-1] and "Drosophila",cite journal |author=Boutros M, Kiger A, Armknecht S, Kerr K, Hild M, Koch B, Haas S, Paro R, Perrimon N |title=Genome-wide RNAi analysis of growth and viability in Drosophila cells |journal=Science |volume=303 |issue=5659 |pages=832–5 |year=2004 |pmid=14764878 |doi=10.1126/science.1091266] 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.cite journal |author=Fortunato A, Fraser A |title=Uncover genetic interactions in "Caenorhabditis elegans" by RNA interference |journal=Biosci Rep |volume=25 |issue=5–6 |pages=299–307 |year=2005 |pmid=16307378 |doi=10.1007/s10540-005-2892-7] 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.cite journal |author=Cullen L, Arndt G |title=Genome-wide screening for gene function using RNAi in mammalian cells |journal=Immunol Cell Biol |volume=83 |issue=3 |pages=217–23 |year=2005 |pmid=15877598 |doi=10.1111/j.1440-1711.2005.01332.x]

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 librariescite journal |author=Huesken D, Lange J, Mickanin C, Weiler J, Asselbergs F, Warner J, Meloon B, Engel S, Rosenberg A, Cohen D, Labow M, Reinhardt M, Natt F, Hall J |title=Design of a genome-wide siRNA library using an artificial neural network |journal=Nat Biotechnol |volume=23 |issue=8 |pages=995–1001 |year=2005 |pmid=16025102 |doi=10.1038/nbt1118] and to predict their likely efficiency at gene knockdown.cite journal |author=Ge G, Wong G, Luo B |title=Prediction of siRNA knockdown efficiency using artificial neural network models |journal=Biochem Biophys Res Commun |volume=336 |issue=2 |pages=723–8 |year=2005 |pmid=16153609 |doi=10.1016/j.bbrc.2005.08.147] 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.cite journal |author=Janitz M, Vanhecke D, Lehrach H |title=High-throughput RNA interference in functional genomics |journal=Handb Exp Pharmacol |volume= |issue= |pages=97–104 |year=2006 |pmid=16594612] cite journal |author=Vanhecke D, Janitz M |title=Functional genomics using high-throughput RNA interference |journal=Drug Discov Today |volume=10 |issue=3 |pages=205–12 |year=2005 |pmid=15708535 |doi=10.1016/S1359-6446(04)03352-5] 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.cite journal |author=Geldhof P, Murray L, Couthier A, Gilleard J, McLauchlan G, Knox D, Britton C |title=Testing the efficacy of RNA interference in Haemonchus contortus |journal=Int J Parasitol |volume=36 |issue=7 |pages=801–10 |year=2006 |pmid=16469321 |doi=10.1016/j.ijpara.2005.12.004] cite journal |author=Geldhof P, Visser A, Clark D, Saunders G, Britton C, Gilleard J, Berriman M, Knox D. |title=RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects |journal=Parasitology |volume= 134|issue= |pages=1–11 |year=2007 |pmid=17201997 |doi=10.1017/S0031182006002071]

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)cite journal |author=Travella S, Klimm T, Keller B |title=RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat |journal=Plant Physiol |volume=142 |issue=1 |pages=6–20 |year=2006 |pmid=16861570 |doi=10.1104/pp.106.084517] as well as more common plant model systems "Arabidopsis" and maize.cite journal |author=McGinnis K, Chandler V, Cone K, Kaeppler H, Kaeppler S, Kerschen A, Pikaard C, Richards E, Sidorenko L, Smith T, Springer N, Wulan T |title=Transgene-induced RNA interference as a tool for plant functional genomics |journal=Methods Enzymol |volume=392 |issue= |pages=1–24 |year=2005 |pmid=15644172 |doi=10.1016/S0076-6879(04)92001-0]

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.cite journal |author=Paddison P, Caudy A, Hannon G |title=Stable suppression of gene expression by RNAi in mammalian cells |journal=Proc Natl Acad Sci USA |volume=99 |issue=3 |pages=1443–8 |year=2002 |pmid=11818553 |doi=10.1073/pnas.032652399] Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus,cite journal |author=Sah D |title=Therapeutic potential of RNA interference for neurological disorders |journal=Life Sci |volume=79 |issue=19 |pages=1773–80 |year=2006 |pmid=16815477 |doi=10.1016/j.lfs.2006.06.011 ] RNAi has also been shown to be effective in the reversal of induced liver failure in mouse models.cite journal |author=Zender L, Hutker S, Liedtke C, Tillmann H, Zender S, Mundt B, Waltemathe M, Gosling T, Flemming P, Malek N, Trautwein C, Manns M, Kuhnel F, Kubicka S |title=Caspase 8 small interfering RNA prevents acute liver failure in mice |journal=Proc Natl Acad Sci USA |volume=100 |issue=13 |pages=7797–802 |year=2003 |pmid=12810955 |doi=10.1073/pnas.1330920100]

Other proposed clinical uses center on antiviral therapies, including the inhibition of viral gene expression in cancerous cells,cite journal |author=Jiang M, Milner J |title=Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference |journal=Oncogene |volume=21 |issue=39 |pages=6041–8 |year=2002 |pmid=12203116 |doi=10.1038/sj.onc.1205878] knockdown of host receptors and coreceptors for HIV, [cite journal |author=Crowe S |title=Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication, by Martínez et al |journal=AIDS |volume=17 Suppl 4 |pages=S103–5 |year=2003 |pmid=15080188 |url=http://www.medscape.com/viewarticle/467320] the silencing of hepatitis Acite journal |author=Kusov Y, Kanda T, Palmenberg A, Sgro J, Gauss-Müller V |title=Silencing of hepatitis A virus infection by small interfering RNAs |journal=J Virol |volume=80 |issue=11 |pages=5599–610 |year=2006 |pmid=16699041 |doi=10.1128/JVI.01773-05] and hepatitis B genes,cite journal |author=Jia F, Zhang Y, Liu C |title=A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference |journal=Biotechnol Lett |volume=28 |issue=20 |pages=1679–85 |year=2006 |pmid=16900331 |doi=10.1007/s10529-006-9138-z] silencing of influenza gene expression,cite journal |author=Li Y, Kong L, Cheng B, Li K |title=Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus |journal=Avian Dis |volume=49 |issue=4 |pages=562–73 |year=2005 |pmid=16405000 |doi=10.1637/7365-041205R2.1] and inhibition of measles viral replication.cite journal |author=Hu L, Wang Z, Hu C, Liu X, Yao L, Li W, Qi Y |title=Inhibition of Measles virus multiplication in cell culture by RNA interference |journal=Acta Virol |volume=49 |issue=4 |pages=227–34 |year=2005 |pmid=16402679] Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease.cite journal |author=Raoul C, Barker S, Aebischer P |title=Viral-based modelling and correction of neurodegenerative diseases by RNA interference |journal=Gene Ther |volume=13 |issue=6 |pages=487–95 |year=2006 |pmid=16319945 |doi=10.1038/sj.gt.3302690] 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.cite journal |author=Putral L, Gu W, McMillan N |title=RNA interference for the treatment of cancer |journal=Drug News Perspect |volume=19 |issue=6 |pages=317–24 |year=2006 |pmid=16971967 |doi=10.1358/dnp.2006.19.6.985937] cite journal |author=Izquierdo M |title=Short interfering RNAs as a tool for cancer gene therapy |journal=Cancer Gene Ther |volume=12 |issue=3 |pages=217–27 |year=2005 |pmid=15550938 |doi=10.1038/sj.cgt.7700791] 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.cite journal |author=Li C, Parker A, Menocal E, Xiang S, Borodyansky L, Fruehauf J |title=Delivery of RNA interference |journal=Cell Cycle |volume=5 |issue=18 |pages=2103–9 |year=2006 |pmid=16940756] cite journal |author=Takeshita F, Ochiya T |title=Therapeutic potential of RNA interference against cancer |journal=Cancer Sci |volume=97 |issue=8 |pages=689–96 |year=2006 |pmid=16863503 |doi=10.1111/j.1349-7006.2006.00234.x]

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.cite journal |author=Tong A, Zhang Y, Nemunaitis J |title=Small interfering RNA for experimental cancer therapy |journal=Curr Opin Mol Ther |volume=7 |issue=2 |pages=114–24 |year=2005 |pmid=15844618] A computational genomics study estimated that the error rate of off-target interactions is about 10%. One major study of liver disease in mice led to high death rates in the experimental animals, suggested by researchers to be the result of "oversaturation" of the dsRNA pathway,cite journal |author=Grimm D, Streetz K, Jopling C, Storm T, Pandey K, Davis C, Marion P, Salazar F, Kay M |title=Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways |journal=Nature |volume=441 |issue=7092 |pages=537–41 |year=2006 |pmid=16724069 |doi=10.1038/nature04791] 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.

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.cite journal |author=Sunilkumar G, Campbell L, Puckhaber L, Stipanovic R, Rathore K |title=Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol |journal=Proc Natl Acad Sci USA |volume=103 |issue=48 |pages=18054–9 |year=2006 |pmid=17110445 |doi=10.1073/pnas.0605389103] Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.cite journal |author=Siritunga D, Sayre R |title=Generation of cyanogen-free transgenic cassava |journal=Planta |volume=217 |issue=3 |pages=367–73 |year=2003 |pmid=14520563 |doi=10.1007/s00425-003-1005-8]

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 plantscite journal |author=Le L, Lorenz Y, Scheurer S, Fötisch K, Enrique E, Bartra J, Biemelt S, Vieths S, Sonnewald U |title=Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression |journal=Plant Biotechnol J |volume=4 |issue=2 |pages=231–42 |year=2006 |pmid=17177799 |doi=10.1111/j.1467-7652.2005.00175.x] and decreased the precursors of likely carcinogens in tobacco plants.cite journal |author=Gavilano L, Coleman N, Burnley L, Bowman M, Kalengamaliro N, Hayes A, Bush L, Siminszky B |title=Genetic engineering of Nicotiana tabacum for reduced nornicotine content |journal=J Agric Food Chem |volume=54 |issue=24 |pages=9071–8 |year=2006 |pmid=17117792 |doi=10.1021/jf0610458] Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy,cite journal |author=Allen R, Millgate A, Chitty J, Thisleton J, Miller J, Fist A, Gerlach W, Larkin P |title=RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy |journal=Nat Biotechnol |volume=22 |issue=12 |pages=1559–66 |year=2004 |pmid=15543134 |doi=10.1038/nbt1033] resistance to common plant viruses,cite journal |author=Zadeh A, Foster G |title=Transgenic resistance to tobacco ringspot virus |journal=Acta Virol |volume=48 |issue=3 |pages=145–52 |year=2004 |pmid=15595207] and fortification of plants such as tomatoes with dietary antioxidants.cite journal |author=Niggeweg R, Michael A, Martin C |title=Engineering plants with increased levels of the antioxidant chlorogenic acid |journal=Nat Biotechnol |volume=22 |issue=6 |pages=746–54 |year=2004 |pmid=15107863 |doi=10.1038/nbt966] 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.cite journal |author=Sanders R, Hiatt W |title=Tomato transgene structure and silencing |journal=Nat Biotechnol |volume=23 |issue=3 |pages=287–9 |year=2005 |pmid=15765076 |doi=10.1038/nbt0305-287b] cite journal |author=Chiang C, Wang J, Jan F, Yeh S, Gonsalves D |title=Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya |journal=J Gen Virol |volume=82 |issue=Pt 11 |pages=2827–36 |year=2001 |pmid=11602796]

History and discovery

The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants,cite journal |author=Ecker JR, Davis RW |title=Inhibition of gene expression in plant cells by expression of antisense RNA |journal=Proc Natl Acad Sci USA |volume=83 |issue=15 |pages=5372–5376 |year=1986 | doi= 10.1073/pnas.83.15.5372 | pmid=16593734] 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.cite journal |author=Napoli C, Lemieux C, Jorgensen R |title=Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans |journal=Plant Cell |volume=2 |issue=4 |pages=279–289 |year=1990 |pmid=12354959 |doi=10.1105/tpc.2.4.279] 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",cite journal |author=Romano N, Macino G |title=Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences |journal=Mol Microbiol |volume=6 |issue=22 |pages=3343–53 |year=1992 |pmid=1484489 |doi=10.1111/j.1365-2958.1992.tb02202.x] 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.cite journal | author = Van Blokland R, Van der Geest N, Mol JNM, Kooter JM | title = Transgene-mediated suppression of chalcone synthase expression in "Petunia hybrida" results from an increase in RNA turnover | journal = Plant J | year = 1994 | volume = 6 | pages = 861–77 | url=http://www.blackwell-synergy.com/links/doi/10.1046/j.1365-313X.1994.6060861.x/abs/ | doi = 10.1046/j.1365-313X.1994.6060861.x/abs/ ] This phenomenon was called "co-suppression of gene expression", but the molecular mechanism remained unknown. [cite book | title=Antisense nucleic acids and proteins: fundamentals and applications | author=Mol JNM, van der Krol AR| year=1991| pages=4, 136| publisher=M. Dekker| isbn=0824785169]

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.cite journal | author = Covey S, Al-Kaff N, Lángara A, Turner D | title = Plants combat infection by gene silencing | journal = Nature | year = 1997 | volume = 385 | pages = 781–2 | doi= 10.1038/385781a0 ] 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.cite journal | author = Ratcliff F, Harrison B, Baulcombe D| title = A Similarity Between Viral Defense and Gene Silencing in Plants | journal = Science | year = 1997 | volume = 276 | pages = 1558–60 | doi= 10.1126/science.276.5318.1558 ]

After these initial observations in plants, many laboratories around the world searched for the occurrence of this phenomenon in other organisms.cite journal |author=Guo S, Kemphues K |title=par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed |journal=Cell |volume=81 |issue=4 |pages=611–20 |year=1995 |pmid=7758115 |doi=10.1016/0092-8674(95)90082-9] cite journal |author=Pal-Bhadra M, Bhadra U, Birchler J |title=Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by white-Adh transgenes is Polycomb dependent |journal=Cell |volume=90 |issue=3 |pages=479–90 |year=1997 |pmid=9267028 |doi=10.1016/S0092-8674(00)80508-5] Craig C. Mello and Andrew Fire's 1998 "Nature" paper reported a potent gene silencing effect after injecting double stranded RNA into "C. elegans". In investigating the regulation of muscle protein production, they observed that neither mRNA nor antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term "RNAi". Fire and Mello's discovery was particularly notable because it represented the first identification of the causative agent for the phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work.

References

External links

* [http://www.nature.com/focus/rnai/animations/animation/animation.htm Animation of the RNAi process] , from "Nature"
* [http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html NOVA scienceNOW explains RNAi] – A 15 minute video of the "Nova" broadcast that aired on PBS, July 26, 2005
*PLoSlink
link=http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371/journal.pbio.0020133
title=Planting the Seeds of a New Paradigm

* [http://www.silencinggenomes.org Silencing Genomes] RNA interference (RNAi) experiments and bioinformatics in C. elegans for education. From the Dolan DNA Learning Center of Cold Spring Harbor Laboratory.
* [http://www.natureprotocols.com/2006/07/19/rnai_screens_in_c_elegans_in_a.php RNAi screens in C. elegans in a 96-well liquid format and their application to the systematic identification of genetic interactions (a protocol)]
* [http://www.nytimes.com/2006/10/03/science/03nobel.html?ex=1180065600&en=e26ec371cb398ea0&ei=5070 2 American ‘Worm People’ Win Nobel for RNA Work] , from NY Times"
* [http://www.nature.com/mt/webfocus/rnai/index.html#2008 "Molecular Therapy" web focus: "The development of RNAi as a therapeutic strategy" ] , a collection of free articles about RNAi as a therapeutic strategy.


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