Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:869±882 (2000)

ReviewPost-transcriptional gene-silencing and RNAinterference: genetic immunity, mechanismsand applicationsMark Turner* and Wolfgang SchuchZeneca Agrochemicals, Jealott’s Hill, Bracknell, Berkshire, RG42 6ET, UK

(Rec

* CoE-maCont

# 2

Abstract: This review summarises the development of our understanding of co-suppression in plants,

and describes how co-suppression relates to similar post-transcriptional gene-silencing (PTGS)

phenomena, and underlying genetic immunity mechanisms. These comparisons have enabled us to

develop sophisticated models that go some way to explaining what causes co-suppression, and indicate

that manipulation of the underlying mechanisms can provide the plant biotechnologist with new tools

to control gene expression and silencing in transgenic plants. Recently, it has become apparent that the

mechanism underlying PTGS has, at least in essence, been conserved through divergent evolution and

similar mechanisms to co-suppression are functional in nematode, insect and mammalian cells. In

addition to the original applications in plant biotechnology, the development of technology based on

PTGS is proving to be a highly valuable alternative to mutagenesis for functional genomics

applications in both plant and animal model species. The recent discovery that PTGS mechanisms

operate in mammalian cells indicates that there are likely to be wider applications for technology based

on PTGS, possibly including human therapeutics.

# 2000 Society of Chemical Industry

Keywords: gene-silencing; RNAi; co-suppression; antisense; functional genomics; genetic immunity

NOTATIONaRNA Aberrant RNA

eived 2

rresponil: marract/gra

000 So

asRNA Antisense RNA

cDNA Complementary DNA

cRNA Complementary RNA

dsRNA Double-stranded RNA

GFP Green ¯uorescent protein

HdRT Homology-dependent RNA-turnover

IR Invert-repeat

mRNA Messenger RNA

nt Nucleotide

PTGS Post-transcriptional gene-silencing

PVX Potato virus X

RdRP RNA-dependent RNA polymerase

RIGS Repeat-induced gene-silencing

RISC RNA-induced silencing complex

RNAi RNA interference

RT-PCR Reverse-transcriptase polymerase chain

reaction

SIGS Systemic-induced gene-silencing

ssDNA Single-stranded DNA

UTR Untranslated region

5 April 2000; accepted 9 May 2000)

dence to: Mark Turner, Zeneca Agrochemicals, Jealott’s Hill, Bk.turner@aguk.zeneca.comnt sponsor: EU; contract/grant number: BI04-96-0253

ciety of Chemical Industry. J Chem Technol Biotechnol 02

1 INTRODUCTION1.1 Genetic and epigenetic gene-regulationGenetic mutation has had a major impact in both

fundamental and applied biology. It is the underlying

mechanism driving natural selection, a powerful tool

in the study of gene-function and also a major

contributing factor or underlying cause of several

diseases. The advent of recombinant DNA technology

and genome sequencing has allowed us to explore

genetic mutation at the DNA sequence level, and this

has led to the wide exploitation of mutagenesis as a

tool in functional genomics. However, there are

numerous examples described in the literature where

phenotypic mutation arises without mutation of the

DNA sequence, and these are described as epigenetic.

We are now beginning to understand the molecular

basis for epigenetic phenomena and perhaps not

surprisingly this is providing us with a considerable

insight into the fundamental mechanisms regulating

gene-expression, development and genetic immunity.

The role of these epigenetic mechanisms has major

implications both for the study of gene expression and

racknell, Berkshire, RG42 6ET, UK

68±2575/2000/$30.00 869

Figure 1. Genome immunity. Repeated DNA sequences are oftenmethylated (M) and exist in dense chromatin structures known asnucleosomes. DNA methylation may be targeted to sequences within thedense nucleosome structure (a). Alternatively, repeated sequences couldbe recognised and methylated de novo, the methylated DNA couldsubsequently be recognised by methylated DNA binding proteins (B),which in turn are known to recruit histone deacetylase complexes (H).Histone deacetylation results in nucleosome formation (b) andtranscriptional inactivation. In order for cell division to occur thenucleosome needs to be disassembled. DNA methylation patterns can beinherited through mitosis and meiosis, allowing for maintenance of DNAmethylation patterns in cell progeny and re-targeting of repetitive DNAsequences for nucelosome formation and transcriptional inactivation (c).

M Turner, W Schuch

function, as well as plant and medical biotechnology.

In addition, it is now widely recognised that epigenetic

disorders can be the underlying cause of serious

diseases, including some forms of cancer.1

In most epigenetic phenomena DNA methylation

and/or chromatin formation are involved. DNA

methylation is a chemical modi®cation of DNA, which

usually occurs at cytosine residues. Chromatin de-

scribes the densely packed DNA and histone protein

complex that forms the nucleosome structure. Both

cytosine methylation and chromatin are heavily

implicated in the control of cell differentiation and

developmental regulation via repression of gene-

expression.2±5 However, it now appears that these

repression mechanisms have also evolved a critical

function in maintaining the integrity of the genetic

material by selective inactivation of invasive or

parasitic nucleic acids.6 The function of this genetic

immunity mechanism could have major implications

on the future success gene-therapy,7 and has been of

major concern for the commercial application of plant

biotechnology.8

1.2 Gene-silencing and genetic immunityLarge regions of mammalian and plant genomes are

comprised of repetitive DNA sequences which are

hypermethylated, and packed into dense chromatin

structures known as nucleosomes, which are tran-

scriptionally inactive. Such sequences have arisen

following the invasion of the genome by retroviruses

and transposable elements, and that DNA methylation

and chromatin have evolved a function to restrict

activities of such parasitic DNA.9,10 There are some

species, including Drosophila and yeast, which lack the

ability to methylate their DNA, indicating that DNA

methylation may not be the primary mechanism

underlying gene-silencing. The discovery that methy-

lated DNA binding proteins can mediate the forma-

tion of chromatin structure,11 and the requirement of

nucleosome formation for gene-inactivation of methy-

lated DNA templates,12 provides substantial evidence

that DNA methylation per se may not be capable of

causing gene-inactivation, but acts as a genetic label to

recruit histone deacetylases, the enzymes that mediate

the packing of genomic DNA into nucleosomes (Fig

1(a and b)). The nucleosome structure may make the

underlying DNA be inaccessible to transcription

factors to the underlying DNA and prevent the

binding and progression of RNA polymeraseII. Such

a mechanism of labelling invasive and repetitive DNA

for silencing would allow for the disassembly of

nucleosomes during DNA replication, and by faithful

replication of DNA methylation patterns the targeted

silencing of parasitic DNA in the progeny cells (Fig

1(c)). How repetitive DNA is targeted for chromatin-

mediated repression in species which lack the ability to

methylate their DNA is unclear, and indicates that

there may be alternative mechanisms involved which

we have not yet discovered.

Current technologies deployed in the introduction

870

of transgenes into plant genomes cause random

insertions and often result in the insertion of several

copies of the transgene at a single locus, or insertion

within or near an area of inactive, chromatin-repressed

DNA. If transgenes insert into transcriptionally-

repressed loci, the repression can spread and inactivate

the transgene (known as cis-inactivation, Fig 2(a)).

The expression of transgenes can also be effected by

the developmental regulation of chromatin-mediated

repression, a phenomenon known as position effect

variegation or PEV.13 The insertion of multiple

transgenes can lead to transgene-inactivation via the

genetic immunity mechanisms that silence repetitive

sequences (Fig 2(b)). Such events can also effect the

silencing of unlinked and genetically distant homo-

logous transgene loci ie trans-inactivation (Fig 2(c)). It

seems somewhat implausible that distant genetic loci

can interact directly, and there is mounting evidence

that an RNA species may mediate trans-inactivation as

a key feature of genetic immunity.14,15 When homol-

ogy occurs between the transgene loci and an

endogenous plant gene loci trans-inactivation can

result in the silencing of both genes, a phenomenon

known as co-suppression (Fig 2(d)).

2 CO-SUPPRESSIONCo-suppression was ®rst encountered in plants during

attempts to over-express endogenous genes by the

introduction of sense constructs (Plate 1). In some

cases this resulted in the silencing of both the

endogenous gene and the transgene, ie co-suppres-

J Chem Technol Biotechnol 75:869±882 (2000)

Figure 2. (a and b ): Cis-inactivation. During plant transformationtransgenes are inserted in a random fashion within the nuclear genome.Insertion can occur into repetitive and transcriptionally repressed loci, andthis repression can spread into the transgene, resulting in silencing (a). Thisis known as a position effect. Alternatively, several transgenes can insert atthe same loci, resulting in repetitive DNA sequences which can beinactivated without a position effect (b). (c and d ): Trans-inactivation.Silenced transgenes can act in trans and inactivate distant, geneticallyunlinked loci that share homology. Trans-inactivation can occur betweenhomologous transgene loci (c), or between a transgene and an endogene,a phenomenon known as co-suppression (d). Trans-inactivation is usuallycaused by complex transgene loci, and can direct alteration of DNAmethylation (M) between homologous sequences (c), and results inalteration of epigenetic state.

Figure 3. The RNA threshold hypothesis. In its simplest form the RNAthreshold describes a hypothesis where the over-expression of a plantgene by the introduction of a transgene results in the accumulation ofmRNA, which can be recognised by the plant cell, triggering of thehomology-dependent RNA-turnover (HdRT) mechanism which results ingene-silencing.

Post-transcriptional gene-silencing and RNA interference

sion.16±19 It is possible that co-suppression can occur

when homology exists within the untranscribed

regions. However, the targeted transcriptional inacti-

vation of the regulatory regions of an endogenous gene

has yet to be demonstrated. All the examples of co-

suppression that have been described in plants are

forms of post-transcriptional gene silencing (PTGS),

and exhibit highly ef®cient homology-dependent RNA

turnover.20±22

Co-suppression is usually only found in a proportion

of transgenic plants, and in some cases only speci®c

branches or sectors show the silencing phenotype.23 At

the time of its discovery, this unpredictable nature of

co-suppression had many consequences: to the scien-

tist it was an intriguing example of epigenetic gene-

regulation, but it also cast doubt over the future

application of transgenic plants in biotechnology.8,24

Co-suppression has also provided plant biotechnology

with a tool to speci®cally target down-regulation of

endogenous genes to enhance crop characteristics18

(Plate 2) and is also of major value as a highly effective

gene-knock out tool for use in functional genomics.25

The ®rst reports of co-suppression were published

over 10 years ago and we are beginning to understand

only now the epigenetic mechanisms underlying the

co-suppression phenomenon, and what features of the

plant transformation process lead to co-suppression.

In addition to the study of co-suppressed plants

J Chem Technol Biotechnol 75:869±882 (2000)

themselves, our knowledge of the mechanism involved

has largely come from the remarkable similarities of

co-suppression to other examples of PTGS, including

RNAi in Caenorhabditis elegans and Drosophila, plant

virus resistance mechanisms, and recently the identi-

®cation of the genes involved in co-suppression and

their conservation between plants, fungi (Neurospora)

and C elegans.26

3 RNA THRESHOLD MODELIt was apparent from early plant transformation

experiments that there was little or no correlation

between copy number and transgene-expression level

and that co-suppression correlates with the insertion of

multiple transgene copies.19 However, it was clear that

single copy transgenes can also trigger co-suppression

especially when in the homozygous state.27 Subse-

quently, it was shown that the probability of silencing

can be correlated to the strength of promoter used and

the stability of the mRNA.28 Together these results

indicate that the accumulation of transgene mRNA

beyond a threshold may be a trigger for co-suppression

(Fig 3).

Co-suppression induced by single copy transgenes

can be alleviated by the inclusion of a tobacco matrix

attachment region (MAR) ¯anking the transgene

construct.29 Matrix attachment regions are AT-rich

regions of DNA which are thought to anchor DNA to

the nuclear matrix and insulate genes from chromatin-

mediated repression.30 Interestingly, the tobacco

MAR did not overcome co-suppression induced by

multi-copy transgenes, indicating that the most

important feature of a single copy transgene locus that

determines its ability to trigger co-suppression is its

insertional position within the genome (position

effect).29 In turn, this indicates that co-suppression

may be a consequence of a high rate of transcription

from a heterochromatic template (Fig 4). How

transgene transcription from such unfavourable geno-

mic positions results in co-suppression is unclear, but

871

Figure 4. Aberrant RNA hypothesis. dsRNA can be produced from atransgene loci either by the action of an endogenous promoter flanking the3' insertion site (a), or by the insertion of an invert-repeated transgenestructure (b). dsRNA is thought to be a reliable trigger for homology-dependent RNA-turnover (HdRT) (d). HdRT can also be the result oftransgene insertion into repressive genomic positions, this could result intransgene methylation (M), or chromatin repression (CR), and theproduction of an as yet unidentified aberrant mRNA. Such an aRNAappears to be an inefficient initiator of HdRT as it is dependent ontransgene transcription rate above a threshold (c). Both dsRNA and aRNAare likely candidates for the substrate for RNA-dependent RNA polymerasewhich make short cRNA species that mediate HdRT by a process ofcleavage and hybridisation to target mRNAs (d).

Figure 5. Plant virus infection can result in dsRNA formation. Most plantviruses have positive single-stranded RNA genomes that replicate via theaction of a viral encoded RNA-dependent RNA polymerase (RdRP), forexample potato virus X (PVX) (i). The RdRP uses the genomic strand tomake a complementary negative strand genome (ii). The RdRP can alsouse this negative strand as a template, which results in production of thegenomic positive strand (iii). This process results in the production ofdsRNA, and in the case of virus-induced gene-silencing can be a reliabletrigger for post-transcriptional gene-silencing (PTGS).

M Turner, W Schuch

due to the high degree of homology required between

transgene and endogene it is likely that a new class of

functional RNA species is involved in transferring a

signal to the cytosol triggering RNA-turnover. Such

RNA have been termed aberrant (aRNA).31 Although

the aberrant RNA has yet to be characterised, the

discovery that both co-suppression and plant virus

resistance operate via the same cytosolic homology-

dependent RNA-turnover mechanism indicates that in

many instances the aRNA is a dsRNA. The produc-

tion of dsRNA could arise from the activity of an

endogenous promoter driving antisense transcription

of the transgene (Fig 4(a)), or from invert-repeated

transgene insertions (Fig 4(b)). Alternatively the

aRNA could be the substrate for an RNA-dependent

RNA polymerase (RdRP) that results in the produc-

tion of short complementary RNA (cRNA, Fig 4).

A suitable candidate RdRP cDNA has been cloned

from tomato.32 The tomato RdRP is induced upon

viroid infection and synthesises complementary

RNA (cRNA) fragments <130b in vitro. Homology

searches and Southern hybridisation indicated that

homologous RdRPs were present in several other

plants as well as in yeast and Caenorhabditis elegans.However, no RdRP homologue has been found in

humans. Further evidence that dsRNA is an effective

inducer of PTGS is the discovery that simultaneous

expression of sense and antisense transgenes can be

used to direct reliable gene-silencing or virus resis-

tance.33

872

4 CO-SUPPRESSION AND VIRUS RESISTANCEMost plant viruses consist of single positive (�ve)

stranded RNA genomes that are replicated by a viral-

encoded RdRP. The function of the RdRP is to ®rst

produce a complementary negative (ÿve) strand RNA

species which is then utilised by the RdRP as a

template to produce the �ve strand genome. This

cycle of virus RNA replication results in the produc-

tion of dsRNA (Fig 5). It appears that such dsRNA

intermediates are detected by plants and trigger a

homology-dependent RNA turnover mechanism simi-

lar to co-suppression.34 The ®rst indication that co-

suppression operated via a mechanism that had

evolved for virus-resistance came from the discovery

that when transgenes expressing viral components

were introduced into plants some lines were found to

be virus resistant.35,36 Subsequently, the use of viral

replicon-based cDNAs as expression vectors (Ampli-

con2) has been shown to result in reliable co-

suppression.37 The Amplicon2 vector is an expression

cassette containing the entire potato virus X (PVX)

cDNA except the coat-protein gene is replaced by a

target endogene (Fig 6(a)). This chimeric PVX cDNA

is then delivered into the plant by a transgenic

approach. The ®rst protein to be made from the

PVX cDNA is the PVX RdRP, which results in the

formation of the dsRNA replication intermediates.

The resultant silencing not only eliminated the PVX

RNA but also the endogenous target RNA. The

inclusion of endogenous plant sequences within

infective viral RNAs (PVX) (Fig 6(b)) is also highly

ef®cient at triggering post-transcriptional gene silen-

cing of plant genes (eg phytoene desaturase) in non-

transgenic Nicotiana benthemiana plants.38 This phe-

nomenon is called virus-induced gene-silencing, or

VIGS and provides a useful tool for reverse genetics for

J Chem Technol Biotechnol 75:869±882 (2000)

Figure 6. Plant viral vectors are useful tools for reliable endogene silencingin plants. The use of potato virus X (PVX) cDNA expression cassettes,known as Amplicon2 (a) as plant transformation vectors can result inreliable endogene silencing in Solanaceae species. The manipulation ofPVX virus RNA to include a portion of target endogene sequence, allowsthe use of virus-induced gene-silencing (VIGS) to confers reliable gene-silencing of the target endogene upon recovery of viral infection (b). VIGSallows rapid determination of gene-knock out phenotype in Nicotianabenthemiana.

Post-transcriptional gene-silencing and RNA interference

gene-knock out.25 The use of VIGS to silence a green

¯uorescent protein (GFP) reporter gene not only

results in PTGS of GFP, but also triggers the

methylation of the GFP transgene, whereas endogene

methylation does not appear to be affected.39 GFP

transgenes can also be silenced by the localised

introduction of homologous DNA sequences (via

agro-in®ltration or biolistic DNA delivery), even if

promoterless constructs are introduced. This not only

causes methylation of the transgene, but remarkably

the silencing spreads from the site of in®ltration,

indicating the existence of a systemic signal. This

phenomenon has been termed systemic-induced gene-

silencing or SIGS.40,41 Apparently SIGS is not

effective against endogenous genes, and this could

relate to the apparent inability of VIGS to methylate

endogenes.

The ®rst evidence that a signal was involved in

PTGS came from the study of nitrate reductase co-

suppression in transgenic tobacco in experiments

where grafted non-silenced transgenic plant scions to

co-suppressed stocks. Remarkably the silencing

phenotype spread to the non-silenced tissues, even if

a section of a wildtype plant was grafted between the

stock and scion.42 This clearly illustrated that plants

use systemic signalling as a method of spreading co-

suppression. The homology requirement for PTGS

was maintained via the systemic signal, indicating that

the signal is probably, at least in part, a nucleic acid

molecule. It has been suggested that systemic signal-

ling could re¯ect the wider use of an `RNA super-

highway' in plants re¯ecting a previously undiscovered

method of long-distance communication regulating

gene-expression.43 It is likely that the systemic signal-

ling mechanism has evolved to protect plants from the

spread of viral infection by the rapid transmission of an

early warning signal to initiate PTGS and viral

immunity. Perhaps the best evidence for a link

between virus resistance and PTGS is the observation

J Chem Technol Biotechnol 75:869±882 (2000)

that several viruses have evolved counter-defence

strategies to suppress the mechanism of PTGS.44

5 ABERRANT RNA AND SYSTEMIC SIGNALLINGThe production of aberrant RNA species which

mediate PTGS is a general feature of current models

for co-suppression. RT-PCR has been used to identify

aberrant RNA species in chalcone synthase (ChsA) co-

suppressed petunia,31 and illustrates that in ChsA co-

suppression there is a complete lack of full length

polyadenylated endogene mRNA, a clear indication of

an alteration in the epigenetic state of the endogene. In

the same study, computer modelling of RNA structure

illustrated that the 3' region of the ChsA endogene

RNA may form an almost perfect invert-repeated

structure. The signi®cance of this prediction is

supported by the ®nding that this 3' region accumu-

lated in co-suppressed white petunia ¯owers. The

authors proposed a model of co-suppression where

complementary sequences with speci®c mRNA struc-

ture preferentially survive RNA turnover, and subse-

quently can mediate homology-dependent RNA-

turnover via undergoing hybridisation and cleavage

cycles with target mRNA. The presence of similar 3'fragments has been shown in other studies of co-

suppression including beta 1,3-glucanase.45 What is

not clear is whether the presence of such aRNAs is the

cause or consequence of RNA degradation, or re¯ects/

mediates alteration in the epigenetic state of the

endogene.

The recent discovery of a short 25nucleotide (nt)

antisense RNA species that occurs speci®cally in co-

suppression, post-transcriptional transgene silencing,

VIGS and systemic-induced silencing, provides a

highly suitable candidate for the systemic signal

molecule.46 What is not apparent is what role such

short antisense molecules play in PTGS. It has been

suggested that it is unlikely that they represent RNA

degradation products due to their antisense nature,

and it is more likely that they are the products of an

RNA-dependent RNA polymerase which subse-

quently directs homology-dependent RNA-turnover,

and which due to their small size are likely to be

transmissible through plasmodesmata. This makes

them highly attractive candidates for the systemic

signal. However, it was also illustrated that in the case

of co-suppression 25nt sense RNAs are also present

and at an equivalent level to the antisense species.46 It

is tempting to suggest a hypothesis where the RdRP

produces short 25nt antisense cRNAs which, in a

similar mechanism to that proposed for ChsA co-

suppression,31 may undergo cleavage/hybridisation

cycles with homologous full length RNA, and mediate

RNase activity. This could result in rapid mRNA-

turnover, but the persistence of the small dsRNAs (Fig

4). Such a mechanism would explain the highly

effective RNA-turnover seen in PTGS.

Although RNA is heavily implicated in the cross talk

between unlinked genetic loci in trans-inactivation

873

M Turner, W Schuch

there is little evidence of such direct cross talk between

endogenes and transgenes in co-suppression. Clearly

RNA can mediate the methylation of nuclear DNA

and such RNA-mediated DNA methylation is asso-

ciated with PTGS of transgenes.14,39 It is therefore

possible that not only does the 25nt antisense RNA

mediate RNA-turnover and systemic signalling, but

also interacts with nuclear genes, potentially altering

their epigenetic state. It is known that RNA:DNA

hybrids are highly stable. Indeed, RNA can form a

triple helical structure with the DNA double strand47

and antisense oligonucleotides can form triple helixes

with dsDNA in the nucleus.48 It is possible that such

DNA:RNA hybrid molecules direct DNA methylation

and/or heterochromatin formation. Such an alteration

in endogene epigenetic state or direct RNA inter-

ference within the DNA could explain the production

of aberrant endogene transcripts.31

6 DNA METHYLATION AND CO-SUPPRESSIONCo-suppression has been correlated to methylation of

the transgene and it has been hypothesised that

methylated transgenes could be the template for the

production of an aberrant RNA that mediates co-

suppression.49 However, in several other studies there

has been no correlation between transgene methyla-

tion,50±53 and it has been suggested that the degree of

transgene methylation is correlated to the chromatin

structure and therefore also the transcriptional activity

of the transgene.54 DNA methylation may therefore be

a secondary effect of co-suppression rather than a

mediator. The apparent confusion over the role of

DNA methylation could be due to the use of

methylation-sensitive restriction enzymes in the iden-

ti®cation of methylated cytosine residues. Using this

method methylation can only be detected at the

speci®c palindromic restriction sites, and it is therefore

quite possible that other methylated sites are not

identi®ed. The use of hypomethylation drugs such as

5-azacytidine can result in alleviation of PTGS of

transgenes indicating that DNA methylation is directly

involved in the maintenance of the PTGS of trans-

genes.55 It has been proposed that methylation can act

as the signal to maintain chromatin repression through

mitotic and meiotic cell division. In the case of co-

suppression it appears that although maintained

during mitosis, there is no transfer of epigenetic state

through the germline, and co-suppression is reset at

meiosis.56 Therefore, it appears likely that in co-

suppression DNA methylation is involved in main-

tenance of suppression once initiated, rather than denovo methylation initiating co-suppression. This is

supported by the observations that the silencing of

a GFP transgene via SIGS triggered methylation

and PTGS of the GFP transgene and transcriptional

inactivation when directed against the CaMV35S

promoter.39 However, in the same study there was

no evidence of methylation of an endogenous rubisco

SSU (rbcS) gene silenced by SIGS. The susceptibility

874

of RNA-mediated DNA methylation may therefore be

a unique feature of transgenes, and may relate to their

genomic position or their structure.

The degree of methylation is related to the structure

of the transgene, with more complex transgene loci

tending to be hypermethylated, transcriptionally in-

active and paradoxically strong inducers of co-sup-

pression.57,58 This phenomena is called repeat-

induced gene-silencing, or RIGS.59

7 INVERT-REPEATS ARE STRONG INDUCERS OFCO-SUPPRESSIONThe detailed study of the structure of transgene

insertions in different petunia transgenic lines found

that where two transgenes had inserted in an inverted

arrangement they were heavily methylated and reliably

caused co-suppression, irrespective of whether the

constructs were sense or antisense, and even if

promoterless constructs were used.57,58 However,

despite the high degree of methylation the invert-

repeat (IR) loci did produce transcripts detectable by

RT-PCR, and given the IR nature of the transgene loci

it is likely that these loci produce dsRNA. Reliable

down-regulation was also seen when using an invert-

repeated region of the ACC-oxidase transcribed region

into a tomato transgene construct demonstrated

reliable co-suppression of the endogenous ACC-

oxidase gene in transgenic tomato, indicating that

the IR phenomenon is not restricted to large transgene

arrangements, but also repeats as short as 78bps.60 In

this study the presence of the mRNA was clearly

demonstrated by RNA-protection assay, again a strong

indication that dsRNA was produced. The study of the

structure of eukaryotic genomes has shown that in

many instances they contain a high proportion of

repetitive sequences, and these sequences are often

remnants of retroviruses of transposable elements, and

as indicated previously gene-silencing has evolved to

inactivate these sequences. Therefore, with hindsight,

it is not surprising that the introduction of repetitive

sequences via plant transformation results in the

silencing of the transgene. What was surprising is that

when the repeats are based on transcribed regions the

mechanism of gene-silencing is post-transcriptional,

illustrating that the cytosolic homology-dependent

RNA-turnover mechanism can be triggered by virus

infection within the cytosol, and also by the introduc-

tion of transgenes into the genome. In both instances

the formation of dsRNA seems to be important in

triggering PTGS. Further evidence of the role of

dsRNA has come from the study of gene-silencing in

other organisms such as the ®lamentous fungus

Neurospora crassa, and the model nematode Caenor-habditis elegans.

8 OTHER EXAMPLES OF PTGS8.1 QuellingA co-suppression-like mechanism, quelling, has been

J Chem Technol Biotechnol 75:869±882 (2000)

Figure 7. Updated model for antisense down-regulation. The introductionof an antisense construct could result in the production of dsRNA, either byhybridisation with the endogene mRNA, from invert-repeat loci, or promoterflanking the insertion site (Fig 4). As antisense down-regulation isunreliable is seems unlikely that the expression of antisense mRNA resultsin the formation of dsRNA after hybridisation with endogene mRNA.However, antisense down-regulation could occur via an aberrant RNAintermediate. If the antisense transgene inserts in a repressive position (M,CR), this could result in the production of an aberrant antisenseRNA(asRNA). If the aberrant asRNA accumulates above a threshold it can thenbe available as a substrate for a RNA-dependent RNA polymerase (RdRP),which produces short sense RNAs, which in turn can mediate homology-dependent RNA-turnover by cleavage hybridisation cycles to antisensemRNA. This process would result in the production of short dsRNAs whichpreferentially survive RNA degradation, and can then mediate HdRTagainst both endogenous sense and transgene antisense mRNAs.

Post-transcriptional gene-silencing and RNA interference

described in Neurospora crassa. Quelling is triggered by

the duplication of coding sequences, and occurs via

enhanced mRNA turnover of the duplicated gene. As

in co-suppression quelling is also mediated by a

diffusible trans-acting molecule.61 Due to the remark-

able similarities between co-suppression and quelling,

Neurospora has been developed as a model species for

determining the genetic basis of PTGS, and has led to

the identi®cation of three genes which, in Neurospora,

are essential for PTGS. A large scale mutagenesis

programme identi®ed three independent mutations

that disrupted quelling, these mutations were called

quelling de®cient (qde).62qde1 was found to encode an

RNA-dependent RNA polymerase (RdRP), with

considerable homology to the tomato RdPP63 and

was the ®rst proof that PTGS involves an RdRP. qde2encodes a protein with homology to a translation

initiation factor.64qde3 encodes a protein with high

homology RecQ helicases.65 The function of RecQ

helicases is in unwinding dsDNA, allowing for the

presentation of single-stranded DNA. In Escherichiacoli they are involved in homologous and illegitimate

recombination, and in humans mutations in RecQhelicase genes BLM and WRN lead to an enhanced

frequency of recombination, and premature ageing

(Werner's syndrome),66 or an enhanced frequency of

cancer (Bloom's syndrome).67 qde3 could allow

DNA:DNA interaction between direct or inverted

repeated sequences, resulting in the induction of

speci®c chromatin conformation and the subsequent

production of an aberrant RNA which mediates PTGS

and interacts directly with homologous sequences at

independent loci.65 Alternatively qde3 could unwind

dsDNA to allow interaction with aRNA inducing an

alteration of the epigenetic state. There are homo-

logues of qde1 and qde3 in Arabidopsis and qde1, qde2and qde3 in C elegans (see below).

8.2 AntisenseThe ®rst examples of post-transcriptional regulation of

gene-expression was the discovery of natural antisense

mechanisms in E coli.68 Subsequently it was found that

antisense strategies could be highly effective for

targeted down-regulation of endogenes, both for use

in transgenic plants/plant biotechnology69,70 and in

medical therapeutics. However, the development of

the antisense approach for commercial use in human

therapeutics has not been easy. A considerable amount

of research and development has now led to the ®rst

therapeutic drugs based on antisense oligonucleotides

and numerous technical challenges have had to be

overcome.71

The use of antisense constructs in plant transforma-

tion usually results in a wide range of expression levels,

with only a small proportion of transgenic plants

showing a high degree of down-regulation. If antisense

and endogene mRNA could form dsRNA it would be

expected that antisense would be a highly reliable

method for down-regulation via post-transcriptional

gene-silencing. Clearly this is not the case. Also, there

J Chem Technol Biotechnol 75:869±882 (2000)

is not always a close correlation between the transcrip-

tional activity of the antisense transgene, indicating

that antisense was not dependent on the titration of

endogene mRNA. Recently the application of anti-

sense constructs in transgenic plants has been re-

examined.58 It appears that in the strongest antisense

down-regulated lines the transgenes have inserted as

an invert-repeat. In the same study it was determined

that in the case of ChsA silencing in petunia ¯owers,

even with highly transcribed transgenes, there is

unlikely to be enough antisense mRNA to ef®ciently

down-regulate the endogenous gene via titration, and

it seems likely that as in co-suppression, antisense

down-regulation is mediated via an aberrant RNA.

Again the ability of antisense invert-repeats indicates

that one aberrant feature of the aberrant RNA is that it

can be double stranded, however other aberrant RNA

species may also be involved. These observations

indicate that a common mechanism linking sense and

antisense down-regulation can be explained via the

proposed model of homology-dependent RNA-turn-

over mediated by short cRNA or dsRNA (Fig 7).

8.3 RNA Interference (RNAi)RNAi has been developed as a functional genomics

tool in Caenorhabditis elegans. C elegans is widely used

as a model animal species, particularly in the study of

animal development. The complete genome sequence

is available.72 RNAi involves the introduction of

double-stranded RNA (ie sense and antisense) into

875

M Turner, W Schuch

the nematode either by micro-injection, or via the

introduction of an invert-repeat construct on an

expression plasmid.73,74 The bene®t of introduction

on a plasmid is that the invert-repeat construct is

maintained and inherited, providing a robust pheno-

type. Like the examples of PTGS described above,

RNAi operates via homology-dependent RNA turn-

over, but unlike co-suppression the RNA degradation

functions in both the cytosol and nucleus, and can be

effective against intronic sequences.75 RNAi has also

been described in trypanosomes, planaria, Drosophila,

and zebra ®sh and more recently in immature mouse

embryos.26,76 Several of the genes involved in RNAi

have been cloned from C elegans and two, mut-2 and

mut-7, are also essential for repression of transposition,

implying a direct link between repression of repetitive

DNA sequences and RNAturnover.26 Intriguingly,

mut-7 has homology to both the human WRN RecQand is required for co-suppression.77 Other genes

which are only essential of RNAi include rde1 which

has homologies to large gene family of genes involved

in developmental regulation and germ line main-

tenance including sting, a gene involved in repression

of a repetitive loci in Drosophila.78 However, rde1 has

highest homology to a rabbit translation initiation

factor, eIF2C, and may have a similar function to

qde2.64 Many of the C elegans RNAi mutants are also

involved in transposon silencing, however rde1 mu-

tants are not effected in repression of transposon

mobilisation.78 Co-suppression has been described in

C elegans and several of the RNAi mutations that have

been described are also de®cient in co-suppression.

Surprisingly, rde1 mutants retain the ability to co-

suppress transgenes, unless they are in an invert-repeat

formation, presumably because invert-repeats produce

dsRNA, ie RNAi.

Recently, a study examining RNAi in cultured

Drospophila cells illustrated that RNAi involves an

RNA/protein complex, described as the RNA-induced

silencing complex (RISC).79 The activity of the RISC

initially co-puri®es with ribosomes, and remarkably

after further puri®cation, is closely associated with

25nt sense and antisense RNAs. The requirement for

the 25nt RNA species has obvious parallels with the

discovery of a similar 25nt species in plant PTGS, and

provides excellent evidence that RNAi and co-sup-

pression function, at least in part, via the same

mechanism.

9 MODELS FOR POST-TRANSCRIPTIONAL GENE-SILENCING (PTGS)There have been numerous models proposed for

PTGS.54,80,81 Most models are based on a central

role of the RdRP in production of cRNA, which

mediates homology-dependent RNA-turnover. The

identi®cation of qde1 as an RdRP essential for quelling

in Neurospora provides the conformation of these

hypotheses. Recently, a gene involved in oogenesis in

C elegans, ego-1, has been found to encode an RdRP

876

and is required for silencing of germline-expressed

genes by RNAi.82 It is not clear what the substrate, the

putative aberrant RNA, for the RdRP is (dsRNA/

aRNA of both?), but the 25nt RNA makes a highly

suitable candidate as an RdRP product.46In vitroactivity of the tomato RdRP appears to result in a

range of RNA products ranging from 50 to 130nts.

However, this may re¯ect in vitro activity, or simply

that the PAGE analysis was not targeted to detect

species as small as 25nt.32 The identi®cation of the

RISC provides an excellent candidate for targeted

RNase activity,79 utilising the cRNA products of the

RdRP for directed homology-dependent RNA-turn-

over. The co-precipitation of the RSIC and ribosomes

indicate that the RNA degradation may be ribosome

associated, although it appears unlikely that it is

translation-dependent.60

The homology of rde1 and qde2 to the rabbit

translation initiation factor, eiF2C, is intriguing, and

indicates a possible link between RNAi and translation

initiation. rde1 and qde2 are likely candidates for the

identi®cation of dsRNA/aRNA in the cytosol and

recruitment into a ribosome-associated silencing

complex that includes both the RdRP and RISC (Fig

8a). It would be expected that such a function would

be essential for both RNAi and co-suppression, but

rde1 is not essential for co-suppression.77 This could

implicate rde1 as an important component in the

recognition of dsRNA in RNAi/invert-repeat induced

gene-silencing, but not the putative aRNA species

implicated as the nucleus-cytsol messenger in co-

suppression. Quelling is associated with tandem-

repeated transgene insertions, and not invert-repeats,

therefore qde2 may recognise the putative aRNA

species. This would be consistent with the lack of

rde1 requirement in transposon silencing,77 with the

production of an aRNA, rather than a dsRNA,

mediating both co-suppression, and transcriptional

inactivation.

The recent evidence that an RNA mediates the

trans-inactivation, transcriptional silencing and methy-

lation of unlinked transgene promoters15 illustrates

that the role of aRNA is likely to be essential for both

transcriptional and post-transcriptional trans-inactiva-

tion. The systemic spread of co-suppression is

correlated to both the production of a short dsRNA

and transgene methylation. It is therefore likely that

the short dsRNA can interact directly with nuclear

genes, and mediate an alteration in epigenetic state

that results in production of aRNA (Fig 8b). This in

effect will ensure the rapid spread and ampli®cation of

the signal. Although various models for how RNA

could mediate gene-silencing have been proposed,83

we still have little understanding of what the nature of

such an aRNA is, what feature of the transgene or

transgene:endogene interaction causes its production,

how it interacts with unlinked homologous genetic

loci, the effect of such interactions, and how it triggers

the cytosolic RNA turnover mechanism. It is also likely

that due to the lack of information we are, in some

J Chem Technol Biotechnol 75:869±882 (2000)

Figure 8a. Updated model for post-transcriptional gene-silencing (PTGS):triggering and initiation. PTGS could be triggered by alteration of theepigenetic state of the transgene or endogene following a DNA:DNA orDNA:RNA interaction, possibly mediated by homologues of qde3/mut7 (i),or by the spread of repressive DNA structure into the transgene. Eitherinteraction could result in the production of an aberrant RNA (aRNA). Itappears that aRNA can only trigger PTGS if it accumulates above a specificthreshold, this may relate to export of unstable aRNA into the cytosol.Alternatively PTGS can be triggered by the production of dsRNA, either byan invert-repeated transgene (iii) or by direct introduction via viral infectionor RNA interference (iv). Once in the cytosol dsRNA and aRNA could berecognised by specific translation initiation factor like proteins (rde1/qde2/ego-1 homologues) which target these unusual forms of RNA to plant RNA-dependent RNA polymerase (RdRP/qde1). It seems likely that the RdRPforms part of a ribosome-linked silencing complex, and the product of theRdRP, short 25nt complementary RNAs (asRNA), is directly associatedwith an RNase targeting complex (RNA induced silencing complex, RISC)which utilises the short asRNA to direct homology-dependent RNA-turnover (HdRT). The short asRNA form duplex RNAs with target RNA andsuch short dsRNA species preferentially survive HdRT. It is proposed thatthe short dsRNA move between cells and act as systemic signallingmolecules.

Figure 8b. Spread and maintenance of post-transcriptional gene-silencing(PTGS). Within a cell of a transgenic plant mRNA can be produced fromeither the endogene or transgene (v). The short 25nt dsRNA can arrivewithin the cell via systemic transfer and could be directly recruited by theRNA-induced silencing complex (RISC) which mediates homology-dependent RNA-turnover (vi) and depending on availability of homologousmRNA would amplify the signalling duplex RNA species. It is likely thatrecruitment into the RISC would result in cleavage of the short duplex RNAand, in the absence of target mRNA, could prevent further amplification ofthe signalling molecule and limit the spread of silencing. However, the shortduplex mRNA could enter the nucleus and interact directly with both thetransgene and endogenes, possibly requiring the DNA unwinding activity ofrecQ helicase (qde3/mut7). Such interactions may directly block RNApolymeraseII activity, leading to mis-processed aberrant RNA, or mediateDNA methylation/chromatin formation, resulting in the establishment of analtered epigenetic state. Transcription from such epigentically altered orRNA interfered template could also result in the production of an aberrantRNA and direct the triggering of PTGS (Fig 8a).

Post-transcriptional gene-silencing and RNA interference

cases, drawing conclusions from different forms of

PTGS to attempt to generate an understanding, in the

long run this may cause considerable confusion as

there are likely to be profound differences between

RNAi and co-suppression (eg rde1), and between the

conservation of mechanisms between plants, inverte-

brates and mammals. There is a considerable amount

of research required before we can have answers to all

these questions.

10 APPLICATIONS OF PTGSOver the past 10 years we have developed a consider-

able understanding of post-transcriptional gene-silen-

cing. The discovery that co-suppression is related to

RNAi and the initial characterisation of mutants, and

subsequently the cloning of some of the genes essential

for PTGS have greatly accelerated our understanding

of co-suppression. Over the next few years our

understanding of PTGS and RNAi will be greatly

enhanced by further identi®cation and characterisa-

tion of the genes involved. Plant mutations in PTGS

have been described.85,86 It will be exciting to discover

the function of the genes affected. It is expected that in

several cases the plant genes will have high homology

J Chem Technol Biotechnol 75:869±882 (2000)

and conserved function to those involved in RNAi.

However, the knowledge of the genes affected in

mutants of PTGS will not tell us the whole story. Some

genes may be essential for PTGS, but also for viability

and would not have arisen within mutant collections.

There will also be a requirement for considerable

molecular and biochemical experimentation, such as

those developed using Drosophila extracts.79,87

This development of our understanding in PTGS is

likely to also enhance the current application of gene-

silencing technology, especially in the ®elds of plant

biotechnology, functional genomics and more spec-

ulatively, human therapeutics.

10.1 The impact of gene-silencing on plantbiotechnologyIt is clear by the considerable technical success of the

®rst generation of plant biotechnology products that

co-suppression is not the `Achilles heel' of plant

biotechnology, and there are clearly numerous possi-

ble commercial applications of gene-silencing technol-

ogy in plants.88 Biotechnology companies have

successfully produced highly robust transgenic plants

by a strategy of careful line selection, and have also

exploited co-suppression phenomena to produce the

®rst genetically modi®ed product on sale in the United

Kingdom, the `low polygalacturonase' tomato pureÂe.

Gene-silencing is considered to be the major

limitation to successful over-expression of transgenes

877

M Turner, W Schuch

for bio-processing applications. The production of

plants de®cient in PTGS may allow huge over-

production of high value commodities. For some

applications co-suppression is the favoured outcome,

and we have learnt how to utilise PTGS for reliable

gene-silencing. However, for many crop enhancement

traits, where ectopic gene-repression is required, such

down-regulation strategies for crop improvement are

unlikely to be of value due to the systemic spread of

silencing throughout the plant.

Gene-silencing is still a considerable limitation to

the ef®ciency of production of transgenic plants, and

early identi®cation of multi-copy lines are important

for an ef®cient commercial transformation pipeline. It

is also likely that MARs or other such insulating

elements may also play an important role in preventing

position effect inducing silencing on single copy

transgene loci and therefore will have an impact of

the ef®ciency of the plant biotechnology. However,

some concern still remains about the future use of

multiple transgenes within crop plants.

Clearly, repeated transgene insertions are more

likely to be affected by silencing and this is likely to

impact the strategies for introduction of multiple

(stacked) transgenes within crop germplasm. This will

be of particular concern when transgene regulatory

regions are used repeatedly. There are reports of the

successful use of repeated components, such as the

CaMV35S promoter within the same transformation

vector,88 and success may depend on the speci®c

component used, the distance between repeats, the

insertional position within the genome, or the preven-

tion of pre-insertional recombination within the

transformation vector. There is no ®rm evidence that

silencing is caused by direct interaction of unlinked

transcriptionally competent loci. It appears more likely

that trans-inactivation of a distant loci is the result of

interaction of an aRNA produced from a silenced

transgene. Therefore, the stacking of single loci

transgenes via sexual crossing will probably be more

robust than the introduction of complex transgenes

containing repeated sequences. In the case of co-

suppression, it may be prudent to ensure that

transgenes do not share any homology within the

transcribed regions of other transgenes or endogenes.

The alternative is that gene-stacking could result in the

accumulation of mRNA (or aRNA), from several

transgenes sharing regions of homology, above the

hypothetical threshold and induce silencing. The most

reliable method of ensuring that gene-silencing will

not reduce or eliminate trait ef®cacy will be via the use

of single use gene-control region components. This

may not be possible for multi-gene traits, which rely on

co-suppression ordinated expression of several trans-

genes. In such cases, the use of multiple copies of

promoter sequences may be possible, but multiple use

of leader sequences and 3'untranslated regions

(UTRs) may greatly reduce trait ef®cacy. An alter-

native strategy would be to knock-out the co-suppres-

sion mechanism in plants, but care will have to be

878

taken to ensure there are no detrimental effects in

terms of transposon reactivation, or enhanced suscept-

ibility to viral infection.

The identi®cation of viral components that disrupt

PTGS are of concern for the robustness of co-

suppression applications in genetically modi®ed crops.

However, a greater understanding of how these

components interact with the silencing mechanism

could provide us with useful tools to prevent or control

co-suppression and lead to powerful and novel

biotechnology strategies against plant viral diseases.

10.2 Functional genomicsThe `Genomics Revolution' has provided the tools to

identify genes of interest based on their sequence and

expression pro®les. However, the majority of genes

that have emerged from activities such as the Human

Genome Project do not have a described function.

There is, not surprisingly, a considerable amount of

academic and commercial interest in methods to

rapidly assign speci®c function to previously unchar-

acterised sequences, ie Functional Genomics. Several

approaches involve the production of mutants to

de®ne a gene-knock out phenotype. In plants, a range

of technologies are available for such a purpose,

including genome walking,89 transposon tagging,90

and T-DNA insertional mutagenesis.91 All three

approaches are time-consuming and require a con-

siderable amount of resource to operate at a genomics

scale.

The identi®cation that dsRNA is an powerful

inducer PTGS provides a highly attractive and rapid

method of producing epigenetic mutation to deter-

mine gene-function. This will not replace the use of

conventional mutagenesis for the production of altered

gene-function analysis, but will provide rapid tools for

gene knock with considerable advantage over tradi-

tional gene-knock out strategies. Transgenic ap-

proaches, such as the Amplicon2,37 and invert-

repeat based constructs will be of limited use in target

discovery and validation, as interesting lethal events

would never regenerate from transformation. How-

ever, the Amplicon2 is highly suitable for use as a

cDNA library vector, and is suitable for application in

large-scale random gene-inactivation for non-lethal

phenotype screens. For the identi®cation of lethal

knock-out phenotypes, SIGS could provide an ex-

cellent high-throughput delivery system for both

technologies and would be suitable for high through-

put functional genomics platforms. However, as there

is no evidence to suggest that silencing can spread

from the site of initial inoculation without the presence

of a homologous transgenes it is unlikely that these

technologies will be applicable as a universal func-

tional genomics tool.

In may respects RNAi and VIGS are equivalent

technologies for animals and plants. Both involve

dsRNA induced gene-silencing, and the silencing can

spread from the point of initial introduction. Also, they

have both been developed in model species (C elegans,

J Chem Technol Biotechnol 75:869±882 (2000)

Figure 9. Models for DNA antisense oligonucleotide activity. Single-stranded DNA (ssDNA) oligonucleotides could hybridise withcomplementary target mRNAs and directly block RNA translation (a).Alternatively the ssDNA:mRNA hybrid could be the target for RNase Hactivity which would degrade the mRNA and leave the ssDNAoligonucleotide intact. Given the proposed model for post-transcriptionalgene-silencing (PTGS), it is also possible that antisense oligonucleotidesinteract with the homology-dependent RNA-turnover (HdRT) mechanismthat operates during post-transcriptional gene-silencing (c). This couldresult in the production of a short DNA/RNA hybrid molecule that mediatesHdRT, ensuring rapid and efficient degradation of target mRNA.

Figure 10. Model for the interferon effect in response to the introduction ofdouble-stranded RNA (dsRNA) into mammalian cells. The introduction ofdsRNA into the cell, usually via viral infection, is detected by the dsRNAresponsive protein kinase, which deactivates the translation initiation factor(eiF2C), resulting in a non-specific block on translation, and ultimately celldeath.

Post-transcriptional gene-silencing and RNA interference

Nicotiana benthamiana), but are likely to have much

wider application in other animal and plant species.

Unlike transgenic or mutagenic approaches, VIGS and

RNAi have the added advantage in that lethal knock-

outs can be recorded, and this has clear implications in

terms of their use as target discovery and validation

tools in the agrochemical industry. The development

of invert-repeat based vectors for delivery of RNAi also

allows the introduction of dsRNA in an inducible and

inheritable fashion,74 and the recent exempli®cation of

RNAi in early mouse embryos indicates that RNAi-

based functional genomics could become an important

tool in mammalian cells.

10.3 Therapeutic usesThe development of antisense based therapeutics has

been widely reported to be of major signi®cance in the

future treatment of serious diseases.71 Two mechan-

isms have been proposed for how antisense oligonu-

cleotides function. Once oligonucleotides bind to their

complementary target they can either passively prevent

translation (Fig 9(a)), or alternatively form the

substrate for RNase H, triggering the degradation of

the target mRNA, but retaining the integrity of the

antisense oligonucleotide (Fig 9(b)). As both these

models involve the production of dsRNA it is also

possible that antisense oligonucleotides function in a

J Chem Technol Biotechnol 75:869±882 (2000)

similar fashion to dsRNA, by the formation of an

ssDNA:RNA hybrid that could mediate homology-

dependent RNA-turnover (Fig 9(c)). However, up

until the successful application of dsRNA in mouse

embryonic cells it appeared that RNAi caused non-

speci®c block in translation in mammalian cells,

causing cell death. It is apparent from the literature

that double-stranded viral RNA can trigger cell

death.92,93 This targeted cell death has probably

evolved as an anti-viral mechanism, and occurs via

the activation of a dsRNA-responsive protein kinase,

which phosphorylates and inactivates the translation

initiation factor eIF2a, resulting in a non-speci®c block

on translation. This mechanism is known as the

`interferon effect' (Fig 10). Clearly, the success in

RNAi in mouse embryos illustrates that the interferon

effect is a technical problem for RNAi in early mouse

embryonic cells, and this will undoubtedly concentrate

attention to the robustness of the interferon effect as a

major limitation to RNAi in mammals. It has been

suggested that eiF2A, qde2 and rde1 both recognise

dsRNA and are functionally related proteins.64,78

However in mammalian cells this appears to triggers

cell death rather than RNA degradation, and in that

respect the interferon effect may be a version of RNAi.

If the interferon effect can be overcome how suitable

is RNAi to therapeutic application? RNAi typically

involves long stretches of dsRNA typically 500±

700bps. It is dif®cult to imagine that such large

molecules could provide the systemic delivery required

for successful therapeutics. However, there is evidence

that the 25nt dsRNA is the signal behind the systemic

spread of PTGS in plants and also mediates RNA-

turnover directly. It now seems highly likely that RNAi

and PTGS in plants and fungi operate through the

same fundamental mechanism, and that this mechan-

879

M Turner, W Schuch

ism can operate in mouse cells.76 Although currently

pure speculation, it is not impossible that short dsRNA

could become the basis for successful therapeutic

treatments. Such approaches may be particularly

suited for the treatment of diseases with an epigenetic

basis via RNA mediated re-inactivation of genes or for

viral and retroviral diseases which have vulnerable

cytosolic RNA components to their lifecycle. If current

problems concerning the robustness of gene-therapy

strategies are overcome it is also possible that the use of

invert-repeat based gene-therapy approaches could

also be of future value in speci®c circumstances.94

11 CONCLUSIONSThere is now considerable evidence that nucleic acid

immunity mechanisms protect the genome and cytosol

from the invasion, replication and expression of

parasitic nucleic acids. These mechanisms can operate

at the level of DNA structure by repressing the activity

of repetitive DNA sequences, or at the RNA level by

post-transcriptional gene-silencing by a process known

as homology-dependent RNA-turnover. Both result in

highly effective gene-silencing. The study of co-

suppression and virus resistance in plants has illu-

strated that the DNA and RNA immunity mechanisms

are closely linked, and both can be triggered by

accumulation of aRNA or dsRNA within the cytosol,

or by insertion of repetitive sequences within the

genome. The phenomena of quelling in Neurosporacrassa, and RNA interference (RNAi) in C elegans bear

remarkable similarities to PTGS in plants, and it is

considered highly likely that they re¯ect different

variants of an ancestral genetic immunity mechanism.

The key feature of all PTGS mechanisms described

is the role of dsRNA as a mediator of gene-silencing.

By delivering dsRNA into cells it is clearly possible that

targeted disruption of endogene function can be

achieved, resulting in the provision of powerful tools

which are being exploited for use in functional

genomics and plant biotechnology. The development

of ef®cacious antisense strategies for the treatment of

disease, and the discovery of RNAi operating in

mammalian cells indicates a potential value of dsRNA

in human therapeutics. We are only beginning to

understand the mechanisms involved in PTGS and

other forms of genetic immunity and some, but clearly

not all, of the genes involved have been identi®ed.

Hopefully, continued progress over the next few years

will provide us with a more thorough knowledge of the

mechanisms and allow us to realise the full potential of

technology based on gene-silencing.

ACKNOWLEDGEMENTSThe authors acknowledge support from the EU

(BIO4-96-0253). They also acknowledge the contri-

bution from Roger Hellens (John Innes Centre) who

kindly provided the ®gures of co-suppression in

petunia, and from David Baulcombe (Sainsbury

880

Laboratory) and Plant Bioscience Limited (Norwich,

UK) for information relating to Amplicon2, VIGS

and SIGS.

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