inter. RNA.pdf

insight introduction

Revealing the world of

RNA interference

Craig C. Mello 1,2 & Darryl Conte Jr 2

1 Howard Hughes Medical Institute and 2 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts

01605, USA (e-mail: craig.mello@umassmed.edu)

The recent discoveries of RNA interference and related RNA silencing pathways have revolutionized our

understanding of gene regulation. RNA interference has been used as a research tool to control the

expression of specific genes in numerous experimental organisms and has potential as a therapeutic

strategy to reduce the expression of problem genes. At the heart of RNA interference lies a remarkable

RNA processing mechanism that is now known to underlie many distinct biological phenomena.

a hypothetical stage in the evolution of life some

four billion years ago when RNA may have been

the genetic material and catalyst for emerging life

on Earth 1,2 . This original RNA world, if it ever

existed on Earth, is long gone. But this Insight deals with a

process that reflects an RNA world that is alive and thriving

within our cells — RNA silencing or RNA interference

(RNAi). When exposed to foreign genetic material (RNA or

DNA), many organisms mount highly specific counter

attacks to silence the invading nucleic-acid sequences

before these sequences can integrate into the host genome

or subvert cellular processes. At the heart of these sequence-

directed immunity mechanisms is double-stranded RNA

(dsRNA). Interestingly, dsRNA does more than help to

defend cells against foreign nucleic acids — it also guides

endogenous developmental gene regulation, and can even

control the modification of cellular DNA and associated

chromatin. In some organisms, RNAi signals are trans-

mitted horizontally between cells and, in certain cases,

vertically through the germ line from one generation to the

next. The reviews in this Insight show our progress in under-

standing the mechanisms that underlie RNA-mediated gene

regulation in plants and animals, and detail current efforts

to harness this mechanism as a research tool and potential

therapy. Here we introduce the world of RNAi, and provide

a brief overview of this rapidly growing field.

translation in mammalian cells 4,5 . Second, dsRNA is ener-

getically stable and inherently incapable of further specific

Watson–Crick base pairing. So a model in which dsRNA

activates sequence-specific silencing implies the existence

of cellular mechanisms for unwinding the dsRNA and pro-

moting the search for complementary base-pairing part-

ners among the vast pool of cellular nucleic-acid sequences.

Hypotheses that require a paradigm shift and depend on the

existence of a whole set of hitherto unknown activities are

rarely appealing.

So why was dsRNA proposed as a trigger for RNAi and

why was this idea so rapidly accepted? To answer this question

we must make a brief historical digression. In 1995, Guo and

Kemphues 6 attempted to use RNA complementary to the

C. elegans par-1 mRNA to block par-1 expression. This

technique is known as ‘antisense-mediated silencing’,

whereby large amounts of a nucleic acid whose sequence is

complementary to the target messenger RNA are delivered

into the cytoplasm of a cell. Base pairing between the ‘sense’

mRNA sequence and the complementary ‘antisense’ inter-

fering nucleic acid is thought to passively block the process-

ing or translation of mRNA, or result in the recruitment of

nucleases that promote mRNA destruction 7,8 . To their sur-

prise, Guo and Kemphues found that both the antisense and

the control sense RNA preparations induced silencing. Sense

RNA is identical to the mRNA and so cannot base pair with

the mRNA to cause interference, raising the question of how

this RNA could induce silencing. Was an active silencing

response being triggered against the foreign RNA, regardless

of its polarity? Or was the silencing apparently induced by

sense RNA actually mediated by antisense RNA? (Antisense

RNA was known to contaminate the type of in vitro tran-

scription products used in these assays.) Despite confusion

about the nature of the RNA that triggered the phenomenon,

this so-called antisense-mediated silencing method contin-

ued to be used to silence genes in C. elegans .

More surprises were in store. While using this antisense

technique to silence C. elegans genes, we were amazed to find

that the silencing effect could be transmitted in the germ

line 3 . A remarkably potent silencing signal could be passed

through the sperm or the egg for up to several generations 3,9 .

Equally remarkable, the silencing effect could also spread

from tissue to tissue within the injected animal 3 . Taken

together, the apparent lack of strand specificity, the remark-

able potency of the RNA trigger, and the systemic spread and

inheritance properties of the silencing phenomenon

prompted the creation of a new term, RNAi 10 . Importantly,

the properties of RNAi demanded the existence of cellular

Discovering the trigger

Crucial to understanding a gene-silencing mechanism such

as RNAi is knowing how to trigger it. This is important from

the theoretical perspective of understanding a remarkable

biological response (see review in this issue by Meister and

Tuschl, page 343); but it also has obvious practical ramifica-

tions for using the silencing mechanism as an experimental

tool (see review in this issue by Hannon and Rossi, page

371). The observation by Fire et al . 3 that dsRNA is a potent

trigger for RNAi in the nematode Caenorhabditis elegans

(Fig. 1) was important because it immediately suggested a

simple approach for efficient induction of gene silencing in

C. elegans and other organisms, and accelerated the dis-

covery of a unifying mechanism that underlies a host of

cellular and developmental pathways. However, there were

substantial barriers to the acceptance of the idea that

dsRNA could trigger sequence-specific gene silencing.

First, at the time, dsRNA was thought to be a nonspecific

silencing agent that triggers a general destruction of mes-

senger RNAs and the complete suppression of protein

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T he term ‘RNA world’ was first coined to describe

insight introduction

Te t r a hy m e n a 37 . These and other breakthroughs united previously

disparate fields by identifying a common core mechanism that

involves the processing of dsRNA into small RNA-silencing guides

(Fig. 2). In short, dsRNA had taken the biological world by storm.

Figure 1 RNAi in C . elegans . Silencing of a green fluorescent protein (GFP) reporter in

C . elegans occurs when animals feed on bacteria expressing GFP dsRNA ( a ) but not in

animals that are defective for RNAi ( b ). Note that silencing occurs throughout the body

of the animal, with the exception of a few cells in the tail that express some residual

GFP. The signal is lost in intestinal cells near the tail (arrowhead) as well as near the

head (arrow). The lack of GFP-positive embryos in a (bracketed region) demonstrates

the systemic spread and inheritance of silencing.

Other silencing triggers

Although it was clear that dsRNA was important either as a silencing

trigger or as an intermediate in all the RNAi-related silencing path-

ways, it was not known whether other stimuli (besides dsRNA) could

trigger silencing. For example, silencing in response to a DNA trans-

gene could still involve a dsRNA trigger: the transgene might integrate

itself into the genome in such a way that a nearby promoter, or an

inverted copy of the transgene itself, leads to the production of

dsRNA, which could in turn enter directly into the RNAi pathway.

Consistent with this idea, transgenes engineered to express both sense

and antisense strands of a gene in plants can lead to efficient silencing,

which is more reproducible and robust than that achieved by trans-

genes expressing either strand alone 38 .

But several lines of evidence suggest that transgenes can trigger

silencing through mechanisms not involving a dsRNA trigger (Fig. 2).

A key gene family involved in silencing pathways in plants 39,40 , fungi 41

and C. elegans 42,43 contains genes that encode putative cellular RNA-

dependent RNA polymerases (RdRPs; also known as RDRs). Mem-

bers of this family of proteins were identified in forward genetic

screens — whereby mutant genes are isolated from an organism

showing abnormal phenotypic characteristics — as factors

required for co-suppression in plants and quelling in Neurospora .

(Co-suppression results from post-transcriptional silencing of

both a transgene and the endogenous copies of the corresponding

cellular gene.) Interestingly, although cellular RdRP genes were

required for transgene-mediated co-suppression in plants 39,40 , they

were not essential for virus-induced silencing of a transgene 39 , pre-

sumably because the virus provides its own viral RNA polymerase.

Furthermore, RdRPs have been shown to direct primer-independent

synthesis of complementary RNA 44,45 . Together, these findings suggest

that the transgene or its single-stranded mRNA products could be the

original stimulus for co-suppression and quelling. In this type of

silencing, the RdRP somehow recognizes transgene products as

abnormal or ‘aberrant’ and subsequently converts this initial silencing

trigger into dsRNA 46,47 . In this case, the dsRNA is an intermediate in

the silencing pathway rather than the trigger. The RdRP-derived

dsRNA is then likely to be processed by Dicer and to enter downstream

silencing complexes that are similar, or identical, to those formed in

response to a dsRNA trigger.

But how might the transgene mRNA be recognized as foreign?

The answer to this question is not known. Hence, this ‘aberrant tran-

script’ model has, perhaps undeservedly, received little attention of

late. One possibility discussed by Baulcombe (review in this issue,

page 356) is that high levels of expression of the transgene mRNA

leads to the accumulation of mRNA-processing defects (for example,

non-polyadenylated transcripts) that are somehow recognized by the

RdRP. Alternatively, the transgene DNA or the chromatin itself may

be ‘marked’ for silencing by the cell. When initially delivered to cells,

the transgene DNA could be recognized as foreign owing to its lack of

associated proteins. During the rapid assembly of naked DNA into

chromatin 48 , the host cell may, in self-defence, somehow mark the

transgene chromatin so that RdRP is recruited. RdRP acting on

nascent transcripts could then result in dsRNA formation and subse-

quent silencing. Consistent with this possibility, fission yeast RdRP

was found to physically associate with silent heterochromatin 36 .

Despite the mysterious nature of the silencing mark recognized by

RdRPs, it seems likely that, at least in some cases, RdRPs may produce

dsRNA that functions as an intermediate rather than as the primary

trigger for silencing.

Genetic studies suggest that distinct silencing triggers may also

exist in C. elegans . Both RDE-1 and the dsRNA-binding protein

RDE-4 (ref. 49) are essential for mediating the silencing induced by

mechanisms that initiate and amplify the silencing signal, and led us

to suggest that the RNAi mechanism represents an active organismal

response to foreign RNA 3 .

Although our initial models saw dsRNA as an intermediate in the

amplification of the silencing signal, Fire 3 suggested that dsRNA,

which is often encountered by cells during viral infection, might itself

be the initial trigger. In this model, instead of antisense RNA passively

initiating silencing by pairing with the target mRNA, the presence of

low concentrations of both sense and antisense strands in the RNA

preparation was proposed to result in small amounts of dsRNA: on

introduction into the animal, this dsRNA could be recognized as

foreign, thereby activating cellular amplification and inheritance

mechanisms. Because it was possible to produce and purify in vitro

synthesized RNA and introduce it directly into C. elegans without the

need for transgene-driven expression, this theory was easily tested.

dsRNA proved to be an extremely potent activator of RNAi — at

least 10-fold and perhaps 100-fold more effective than purified

preparations of single-stranded RNA 3 .

Taking the biological world by storm

With the discovery of an extremely potent trigger for RNAi, it became

possible to expose large populations of animals to dsRNA: animals

were soaked in dsRNA 11 or given food containing bacterially

expressed dsRNA 12,13 . By facilitating genetic screens, these methods

led to the identification of many C. elegans genes required for RNAi 14 .

Comparison of the C . elegans genes required for RNAi to genes

required for gene silencing in Drosophila 15,16 , plants 17 and fungi 18

confirmed that the silencing phenomena known variously as post-

transcriptional gene silencing (PTGS) 19 , co-suppression 20 , quelling 21

and RNAi, share a common underlying mechanism that reflects an

ancient origin in a common ancestor of fungi, plants and animals.

This realization was followed by a flurry of exciting results: dsRNA

was shown to induce silencing in Drosophila 22 , and in a host of other

organisms including organisms that were otherwise unsuited to

genetic analysis 23,24 . Small RNAs were shown to be produced in plants

undergoing PTGS 25 , and were identified as the common currency of

RNA silencing pathways 26–28 (see review in this issue by Baulcombe,

page 356). The dsRNA-processing enzyme Dicer 29 was found to pro-

duce these small RNAs, now called short interfering RNAs (siRNAs).

Synthetic RNAs engineered to look like the products of Dicer were

shown to induce sequence-specific gene silencing in human cells

without initiating the nonspecific gene silencing pathways 30 . A class

of natural hairpin dsRNAs 31,32 , now called microRNAs (miRNAs; see

review in this issue by Ambros, page 350), was shown to be processed

by Dicer 33–35 and to function together with RDE-1 homologues 35 ,

thereby linking the RNAi machinery to a natural developmental gene

regulatory mechanism. Finally, more recently, the RNAi machinery

was linked to chromatin regulation in yeast 36 , and to chromosomal

rearrangement during development of the somatic macronucleus in

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Figure 2 Model depicting distinct roles for dsRNA in a network of interacting silencing pathways. In some cases dsRNA functions as the initial stimulus (or trigger), for example

when foreign dsRNA is introduced experimentally. In other cases dsRNA acts as an intermediate, for example when ‘aberrant’ mRNAs are copied by cellular RdRP. Transcription can

produce dsRNA by readthrough from adjacent transcripts, as may occur for repetitive gene families or high-copy arrays (blue dashed arrows). Alternatively, transcription may be

triggered experimentally or developmentally, for example in the expression of short hairpin (shRNA) genes and endogenous hairpin (miRNA) genes. The small RNA products of the

Dicer-mediated dsRNA processing reaction guide distinct protein complexes to their targets. These silencing complexes include the RNA-induced silencing complex (RISC), which

is implicated in mRNA destruction and translational repression, and the RNA-induced transcriptional silencing complex (RITS), which is implicated in chromatin silencing. Sequence

mismatches between a miRNA and its target mRNA lead to translational repression (black solid arrow), whereas near perfect complementarity results in mRNA destruction (black

dashed arrow). Feedback cycles permit an amplification and longterm maintenance of silencing. CH 3 , modified DNA or chromatin; 7mG, 7-methylguanine; AAAA, poly-adenosine

tail; TGA, translation termination codon.

injecting, feeding or expressing dsRNA 14 . However, RDE-1 and RDE-4

are not required for transposon silencing or for co-suppression 14,50,51 .

Furthermore, RDE-1 and RDE-4 are not required for the inheritance

of RNAi-induced silencing 9 , which suggests that they are only required

during the initial exposure to dsRNA. These findings indicate that

transposon silencing and co-suppression in C. elegans are initiated by

means of distinct triggers. As discussed above, an appealing idea is that

a chromatin ‘signature’ stimulates the production of aberrant tran-

scripts and the formation of a novel species of dsRNA (perhaps

nuclear) that is distinct from the dsRNA that initiates silencing by

means of RDE-1 and RDE-4. Again, in this model the initial trigger is

the chromatin structure of the transposon locus or the transgene, and

dsRNA acts as an intermediate in the silencing pathway (Fig. 2). Per-

haps a similar RdRP-derived dsRNA functions in the RDE-1- and

RDE-4-independent mechanisms that propagate silencing from one

generation to the next.

Te n y ears ago, de novo cytosine methylation of genomic DNA was

shown to occur in plants infected with RNA viroids whose sequences

were homologous to the methylated genomic sequences 52 . This

process was referred to as RNA-directed DNA methylation (RdDM).

Subsequently, dsRNA targeting a promoter was shown to trigger

RdDM and initiate transcriptional silencing. The silencing was

accompanied by the production of siRNAs 53 , pointing to an RNAi-

like mechanism for the initiation of transcriptional gene silencing.

Recent work in fission yeast has now convincingly demonstrated that

the formation of silent heterochromatin can be guided by small

RNAs 54 and the RNA-silencing machinery 36 . In Drosophila , the RNA-

silencing machinery was also required for heterochromatin forma-

tion and for silencing multicopy transgenes and pericentric DNA 55 .

The discovery of an underlying molecular connection between RNA

guides and chromatin remodelling has been one of the most exciting

recent developments in the field of epigenetics. It is becoming clear

that RNAi has an important role in the initiation of heterochromatin

formation and transcriptional silencing in plants, fungi and animals

(see review in this issue by Lippman and Martienssen, page 364).

The possibility of feedback between RNAi, its potential chromatin-

associated trigger, and chromatin-mediated silencing maintenance

mechanisms raises further questions about the ultimate causes of

silencing. For example, were C. elegans transposons originally silenced

by means of an RDE-1/RDE-4-dependent dsRNA signal, resulting

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insight introduction

from sense and antisense readthrough transcription from insertion

points in the genome 56,57 ? Perhaps over time this initial dsRNA-

triggered silencing signal was replaced and augmented by a

chromatin-associated silencing-maintenance signal.

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Outlook for the RNA world

The numerous branching and converging silencing pathways that

seem to exist in diverse organisms will no doubt require many years of

research to unravel. It is already clear that different organisms have

evolved distinct mechanisms, or at least variations on a common

theme. In some cases, differences seem to exist in the extent to which

silencing relies on a particular mode of regulation. For example,

plants show a preponderance of miRNA-guided mRNA cleavage 58,59 ,

but only one example of this mode of regulation has been found in

animals 60 . The diversity of RNA silencing phenomena suggests that

other interesting findings await discovery. For example, the existence

of an inheritance mechanism for the transmission of RNAi in C. elegans

raises the question of whether natural small RNAs are transmitted

in germ cells or other developmental cell lineages in other animals,

including humans. Extrachromosomal inheritance of silencing

patterns by means of small RNAs could provide sophisticated layers

of gene regulation, at both post-transcriptional and chromatin-

modifying levels. These small RNAs may be important in stem-cell

maintenance and development, and differential localization of

such RNAs may have a role in the generation of cellular diversity. It

will be interesting to discover if the phenomenon of lateral transport

of RNA from cell to cell, so far observed in plants 61,62 and C. elegans , is

more widespread. As well as having a role in immunity, could

‘epigenetic RNA morphogens’ allow cells to modulate the activity

of developmentally important genes or mRNAs in neighbouring

cells? This type of regulation might be particularly useful when

cells, such as neurons, communicate at junctions that are far from

the cell nucleus.

The past ten years have seen an explosion in the number of non-

coding RNAs found to orchestrate remarkably diverse functions 63,64 .

These functions include: sequence-specific modification of cellular

RNAs guided by small nucleolar RNAs 65 ; induction of chromosome-

wide domains of chromatin condensation by the mammalian non-

coding RNA Xist (X-inactive specific transcript) 66 ; autosomal gene

imprinting and silencing by noncoding mammalian Air (antisense

IgF2r RNA) 67 ; and finally sequence-directed cleavage and/or repression

of target mRNAs and genes by miRNAs and siRNAs, discussed here and

in the accompanying reviews. Some have likened this period to an RNA

revolution. But considering the potential role of RNA as a primordial

biopolymer of life, it is perhaps more apt to call it an RNA ‘revelation’.

RNA is not taking over the cell — it has been in control all along. We just

didn’t realize it until now.

■

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Acknowledgements C.C.M. is an HHMI Assistant Investigator and is funded by the NIH.

D.C. is supported by an NRSA postdoctoral fellowship.

Competing interests statement The authors declare that they have no competing financial

interests.

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