【共享】Nature上一篇非常精彩的RNAI的综述

最近要写一篇关于RNAi的综述，昨天在nature上面找了一篇关于rnai全文的综述，觉得写的挺好，拿出来分享

Nature
(C) 2004 Nature Publishing Group

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Volume 431(7006) 16 September 2004 pp 338-342
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Revealing the world of RNA interference
[Insight: Introduction]
Mello, Craig C.1,2; Conte, Darryl Jr1
1Howard Hughes Medical Institute and 2Program in Molecular Medicine, University
of Massachusetts Medical School, Worcester, Massachusetts 01605, USA (e-mail:
craig.mello@umassmed.edu)

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Outline

Abstract
Discovering the trigger
Taking the biological world by storm
Other silencing triggers
Outlook for the RNA world

Acknowledgements
Competing interests statement

Graphics

Figure 1
Figure 2

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Abstract

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.

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The term 'RNA world' was first coined to describe 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
transmitted 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 understanding 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.

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 ramifications 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 discovery
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.

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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.
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First, at the time, dsRNA was thought to be a nonspecific silencing agent that
triggers a general destruction of messenger RNAs and the complete suppression of
protein translation in mammalian cells 4,5. Second, dsRNA is energetically
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 promoting the
search for complementary base-pairing partners 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'
interfering nucleic acid is thought to passively block the processing or
translation of mRNA, or result in the recruitment of nucleases that promote mRNA
destruction 7,8. To their surprise, 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
transcription products used in these assays.) Despite confusion about the nature
of the RNA that triggered the phenomenon, this so-called antisense-mediated
silencing method continued 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 remarkable 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 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 Drosophila15,16, 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 Drosophila22, 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 produce 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 Tetrahymena37.
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.

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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.
CH3, modified DNA or chromatin; 7mG, 7-methylguanine; AAAA, poly-adenosine tail;
TGA, translation termination codon.
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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 pathways, it was not known
whether other stimuli (besides dsRNA) could trigger silencing. For example,
silencing in response to a DNA transgene 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 transgenes 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. elegans42,43
contains genes that encode putative cellular RNA-dependent RNA polymerases
(RdRPs; also known as RDRs). Members 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, presumably 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 transcript' 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
subsequent 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 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 transcripts 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).
Perhaps a similar RdRP-derived dsRNA functions in the RDE-1- and RDE-4-independent
mechanisms that propagate silencing from one generation to the next.

Ten years 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 formation 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 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.

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 noncoding 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 noncoding 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.

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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