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Biochemical Society Transactions (2004) Volume 32, part 6
Silence is green
A.J. Herr1
Sainsbury Laboratory, John Innes Centre, Norwich, U.K.
Abstract
Small RNAs serve as the specificity determinant for a collection of regulatory mechanisms known as RNA
silencing. Plants use these mechanisms to control the expression of endogenous genes and to suppress
unwanted foreign nucleic acids. Several gene families implicated in silencing have undergone expansion
and evidence exists for multiple RNA silencing pathways. Recent progress in defining the components of a
number of these pathways is examined here.
Introduction
The ability to process double-stranded RNA into small 21–
28 nt fragments by an RNaseIII-based enzyme called Dicer
[or Dicer-like (DCL)] is widespread among eukaryotic cells.
The products of cleavage, termed siRNA (short interfering
RNA) or miRNA (micro-RNA) depending on the nature
of the double-stranded substrate, carry 5 phosphates and
2 nt 3 overhangs, which license them for incorporation into
protein effector complexes that regulate gene expression in
a sequence-specific manner [1,2]. The known evolutionary
diversity speaks of the remarkable number of ways in which
this regulation can occur. Small RNAs have been implicated in
directing endonucleolytic cleavage of mRNA [3], suppressing
protein expression [4], directing double-stranded RNA
synthesis by RDRs (RNA-dependent RNA polymerases) [5],
mediating DNA methylation/heterochromatin formation
[6–8] and even guiding programmed deletion of DNA [9].
Plants are an ideal system to study small-RNA-mediated
regulatory mechanisms because many of the genes implicated
in silencing have undergone amplification and specialization.
Uncovering the various silencing pathways represented by
these gene families is critical for understanding how this
proliferation of genes contributes to plant biology. The
existence of similar non-redundant parallel pathways leads
to the additional question of how much cross-talk occurs
between the different avenues of regulation. Are small RNAs
destined for a particular application from their inception? If
so how is fate enforced? Is there a sharing of small RNA-based
information between different pathways? If so, is this sharing
regulated? Continued characterization of small RNAs in
plants and the genetic requirements for their production/
accumulation are proving useful in defining different silencing
pathways and their potential points of intersection.
From miRNAs to RISC
miRNAs are encoded by dispersed loci within the genome
and arise from precursors (pri-miRNA, primary miRNA),
Key words: Dicer, DNA methylation, miRNA, RNA-induced silencing complex (RISC), siRNA.
Abbreviations used: DCL, Dicer-like; PAZ, Piwi/Argonaute/Zwille; PPD, PAZ and PIWI domain;
PTGS, post-transcriptional gene silencing; RDR, RNA-dependent RNA polymerases; RISC, RNAinduced silencing complex.
1
email [email protected]
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which fold back on themselves to create a partial dsRNA
substrate for a DCL enzyme. Plants have genes for four DCL
proteins (Table 1), each with an N-terminal RNA-helicase
domain followed by tandem RNase III domains (reviewed in
[10]). They also have a PAZ (Piwi/Argonaute/Zwille) domain
in the central part of the protein, which is a standard feature of
animal Dicers (and the signature of the PAZ family of proteins
discussed below) and/or two dsRNA-binding domains at the
C-terminus. DCL1 is required for most, if not all, miRNA
processing as shown by decreased miRNA levels in DCL1
mutants. Null mutations in DCL1 are embryonic lethal [11]
and partial loss of function mutants show severe pleiotropic
defects consistent with misregulation of miRNA-controlled
developmental pathways [12]. A second gene required
for miRNA accumulation is HEN1 (Hua enhancer 1),
which displays overlapping phenotypes with DCL1 mutants
[13].
The DCL1-mediated mechanism of miRNA biogenesis in
plants may differ somewhat from that in flies and mammals.
In these organisms, miRNA formation begins in the nucleus
with Drosha, which contains tandem RNase III domains
but lacks an RNA helicase domain [14,15]. The stem loop
containing the miRNA is excised from the rest of the RNA
and exported to the cytoplasm as a pre-miRNA, where
a second cleavage by Dicer liberates the dsRNA–miRNA
duplex [16]. Subsequent unwinding of the duplex then allows
incorporation of the mature miRNA into a downstream
effector complex as well as degradation of the other strand.
Plants do not encode an equivalent enzyme to Drosha and
DCL1 localizes to the nucleus [17] raising the possibility that
miRNA biogenesis is a one-step process that is completed
before miRNA export from the nucleus.
Export of pre-miRNAs from the nucleus in animal
cells requires exportin-5 [18–20]. Although exportin-5
binds RNA directly, associated proteins can be transported
across the nuclear membrane as well [21–23]. Consistent
with inefficient transport of small RNA regulators from
the nucleus, mutation of the exportin-5 homologue in
plants (HASTY) causes developmental timing defects [24].
However, HASTY is unlikely to be the only transporter of
miRNAs, because the mutants do not display a DCL1-like
phenotype.
Genes: Regulation, Processing and Interference
Table 1 Proven and possible RNA silencing genes in Arabidopsis
Argonautes (ago)
DCL
RDR
Two tandem dsRNA-binding domains
Chromatin level silencing
Others
Gene name
Locus
Function
AGO1
AGO2
At1g48410
At1g31280
AGO3
AGO4
AGO5
At1g31290
At2g27040
At2g27880
Chromatin silencing
AGO6
AGO7/ZIPPY
AGO8
At2g32940
At1g69440
At5g21030
Developmental timing
AGO9
PINHEAD/ZWILLE
DCL1/CAF/SIN1
At5g21150
At5g43810
At1g01040
Meristem identity
miRNA processing
DCL2
DCL3
At3g03300
At3g43920
Viral resistance
Chromatin silencing
DCL4
RDR1
RDR2
At5g20320
At1g14700
At4g11140
Viral defence
Chromatin silencing
RDR3
RDR4
RDR5
At2g19910
At2g19920
At2g19930
RDR6/SDE1/SGS2
HYL1
At2g28380
At3g49500
At1g07900
PTGS viral defence
miRNA biogenesis
At5g41070
At3g62800
MET1
At5g49160
CpG methylation
DDM1
DRM1
DRM2
At5g66750
At1g28330
At1g15380
Chromatin remodelling
CpNpN methylation
CpNpN methylation
CMT3
SGS3/SDE2
SDE3
At1g69770
At5g23570
At1g05460
CpNpG methylation
PTGS viral defence
PTGS viral defence
SDE4
HEN1
At4g20910
PTGS/chromatin silencing
miRNA processing/stability, PTGS, viral defence,
HASTY
At3g05040
chromatin silencing
Exportin 5, miRNA transport?
When liberated from the precursor, plant miRNAs load
into a complex capable of directing endonucleolytic cleavage
[5,25], which is analogous to the RISC (RNA-induced silencing complex) found in animal cells [3,26,27]. Unlike animal
miRNAs, which pair imperfectly with their target RNAs and
suppress protein expression, most plant miRNAs match their
target well and direct mRNA cleavage. miRNA-containing
fractions from animal cells, which suppress protein expression, are capable of directing cleavage if presented with a
homologous target, which suggests that in animal cells
siRNAs and miRNAs feed into at least the same kind of
complex [28,29].
Loading of siRNAs into RISC in Drosophila requires
R2D2, a protein with tandem dsRNA-binding domains,
shown to directly bind siRNAs and to associate with the
Dicer (DCR-2) that generates siRNAs. Arabidopsis HYL1
encodes a nuclear-localized protein with tandem dsRNAbinding domains, produces overlapping dcl1-like phenotypes
when defective and is required for the accumulation of a
number of miRNAs, suggesting that the protein may play
a role analogous to R2D2 in miRNA trafficking [30,31]. If
miRNA processing is nuclear, perhaps HYL1 is shuttled
out of the nucleus along with miRNA. There are other
uncharacterized small proteins with tandem dsRNA-binding
domains in Arabidopsis that may also play a role (Table 1).
Others may function more like RDE4, an R2D2-like essential
component of RNAi in worms, which associates with Dicer
but binds longer dsRNA rather than siRNA duplexes [32].
Animal RISC complexes from flies and human cells require
argonaute [or PPD (PAZ and PIWI domain)] proteins [33,34].
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A series of recent structural studies indicates that the PAZ
domain binds the ends of RNase III cleavage products
[35–38]. The 2 nt 3 overhang is nestled in a binding pocket,
whereas extensive contacts are made with the adjacent seven
nucleotides of that strand [38]. The extent of contacts to
this strand, as well as binding studies to ssRNA, suggests
that the PAZ domain would continue to associate with the
RNA after unwinding of the duplex. In the crystal structure,
minor contacts are also made with a non-phosphorylated 5
nucleotide of the opposite strand [38]. Although the contribution of a phosphorylated 5 nucleotide was not assessed, it
is known from other work to be required for incorporation
into RISC [2].
AGO1, the founding member of the PPD family, was
originally identified in plants from a mutant with pleiotropic
effects on development [39]. Subsequent genetic studies
demonstrated that both severe and hypomorphic ago1 alleles
compromised PTGS (post-transcriptional gene silencing),
in which a single-stranded transcript was converted into
dsRNA [40,41]. A hypomorphic ago1 allele did not affect
silencing triggered by transcription of an inverted repeat,
which suggested that AGO1 acted upstream of DCL [42].
Because Arabidopsis carries ten PPD genes (Table 1), it seemed
possible that AGO1 helped to produce dsRNA while a
different PPD anchored RISC. The only nagging problem
was how to explain the rather severe developmental defects
of strong ago1 alleles. New data now show that these alleles
significantly elevate a number of different miRNA targets
[43]. Functionally, this places AGO1 in plant RISC, but
another interpretation is that AGO1 has a role in directing
miRNA traffic. One line of evidence for this idea is that
the stability of some, but not all, miRNAs is compromised in
strong ago1 alleles. A second line comes from the observation
that in the developing embryo, AGO1 restricts miR165 to
the abaxial surface, where it down-regulates PHABULOSA
and PHAVOLUTA to control abaxial/adaxial patterning [44].
A strong ago1 allele leads to ectopic adaxial expression of
miR165. In spite of increased overlap with its targets, miRNA
is unable to guide mRNA degradation. It is as if miR165
has wandered away from the silencing pathway. The two
ideas are not mutually exclusive and a role for stabilizing
miRNA association with RISC may best explain the influence
of AGO1 in miRNA localization.
If AGO1 acts in RISC, how do inverted repeats silence
genes in ago1 [42]? The ago1 allele used for this test is
hypomorphic and contains residual AGO1-mediated RISC
activity as shown by looking at miRNA target levels [43].
This activity, coupled with the high levels of siRNA derived
from the inverted repeat, is probably sufficient for silencing.
In the PTGS line where silencing was compromised by the
same ago1 mutant, the steady-state siRNA levels may be
naturally lower or the conversion of sense transcripts into
dsRNA and siRNAs may be dependent on AGO1 function.
Thus, a working model of the miRNA pathway in plants
begins with transcription of the pri-miRNA, processing by
DCL1, transport by HYL1, export out of the nucleus and
incorporation into AGO1-containing RISC.
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A network of siRNA-generating pathways
in nucleic acid immunity
siRNAs serve as the specificity determinant of nucleic acid
immunity in plants. They were first discovered in virusinfected plant tissue and from plants undergoing PTGS [45].
Sense and anti-sense RNAs accumulated along the length
of the silenced RNA, implying that long dsRNA from the
virus or transgene was being degraded by a DCL. Northern
blots and direct sequencing of siRNAs revealed two size
classes, centred on 21 and 24 nt [6,46], which appeared to
have different functions in resistance. Intriguingly, viral proteins that block a long-distance sequence-specific silencing
signal that moves through the vasculature also block
production of 24 nt RNA [6].
Analysis of small RNAs from plants demonstrated the
presence of endogenous siRNAs of the same two size
classes [5,46]. Some of the silenced loci produced exclusively
24-nt-long siRNAs [6,47], indicating that the two size classes
represent distinct pathways. Further evidence for two different processing pathways came from wheat-germ extracts,
which produce both size classes of siRNA when seeded
with long dsRNA. Production of a 24 nt RNA, but not a
21 nt RNA, was inhibited in the presence of excess 24 nt ds
siRNA, which is consistent with product inhibition and two
different DCL enzymes [5]. Recent analysis of dcl mutants in
Arabidopsis indicates that DCL3 produces the 24 nt siRNA,
whereas DCL2 produces a 21 nt RNA from virus infections
[47].
In addition to multiple processing pathways there are
multiple routes to dsRNA in plants. One pathway of dsRNA
production was revealed by screens for Arabidopsis mutants
impaired in PTGS [48,49]. In addition to AGO1, mutations
were obtained in the RNA-dependent RNA polymerase
gene, RDR6 (SDE1/SGS2) [48,50], a coiled-coil domain
protein (SGS3/SDE2) [48,50], a DEAD-box RNA helicase
(SDE3) [51], and HEN1 [52]. As with AGO1, these genes
were proposed to be involved in the production of dsRNA
on the basis of two criteria. First, PTGS-specific 21 and 24 nt
siRNAs were lost when these genes were mutant and second,
virus-induced gene silencing or inverted repeat silencing was
still effective [42,48,52].
Plants encode five other RDR genes besides RDR6
(Table 1). The ability to isolate mutations in one implies that
the others may produce dsRNA from different templates. In
keeping with this, RDR1, which is up-regulated in the plant
innate immune response, is required for resistance to TMV
and TRV [53], whereas RDR6 is not [48,50]. No evidence
currently exists regarding the role of RDR3-5 subfamily, but
recent work suggests RDR2 silences endogenous transposons
and other repetitive DNA [47].
The evidence for this chromatin level pathway began
with the finding that SDE4, a gene initially described as
having a partial role in PTGS (Figure 1) [48], was required
for methylation and 24 nt siRNA production from the
retroelement, AtSN1 [6]. Subsequently, AGO4 [54] RDR2,
DCL3 and HEN1 have all been implicated in AtSN1 silencing
Genes: Regulation, Processing and Interference
Figure 1 Silencing of GFP (green fluorescent protein) in sde4-1 is
factor, DDM1, are required for maintenance of PTGS [68]
and for AtSN1 siRNA production [69].
partially compromised
The 4-week-old plant was photographed under UV light. Tissue that is
silenced for GFP appears red due to autofluorescence of chlorophyll.
Cross-talk between pathways
[47]. Additional silenced loci affected by this pathway have
also been identified [47,55], although only a subset requires
HEN1 and A604. Viroids, viruses and certain transgenes
have long been known to direct methylation and silencing
of DNA in plants (termed RNA-directed DNA methylation)
[56–58]. The hallmark of RNA-directed DNA methylation is
methylation of both symmetrical (CpG and CpNpG)
and non-symmetrical cytosines (CpNpN, where N is any
nucleotide). Methylation at CpG and CpNpG sites can be
inherited into the next generation through the action of
the maintenance methyl-transferase (MET1) [59–61] and the
chromodomain containing protein (CMT3) [62] respectively.
Methylation at non-symmetrical sites, however, requires
a continual source of dsRNA and the de novo methyltransferase DRM [63,64].
Consistent with a connection between chromatin and silencing, ago4, rdr2 and dcl3 mutants have all been shown to
shift modification of histone H3 at AtSN1 from a repressed
state to one associated with active chromatin [47,54]. A direct
link between the RNAi machinery and modification of the
histone code has also been shown from work on Schizosaccharomyces pombe. S. pombe Ago, Dicer and RdRp genes
are required for silencing of centromeric repeats [7,8] and
silencing of sequences homologous to inverted repeats
[65,66]. Recently, a complex (termed RITS for RNA-induced
transcriptional gene silencing) has been purified containing
siRNA corresponding to silenced loci, Ago, the chromodomain protein, Chp1 [67], and a novel protein termed Tas3.
This suggests that PPD-based effector complexes may act
like DNA-binding proteins to recruit chromatin-remodelling
complexes to specific loci. Intriguingly, these complexes,
guided by small RNA, may in turn lead to the production
of more small RNA. MET1 and the chromatin-remodelling
An immune system that shares information and amplifies a
defence response has the best chance of eliminating a pathogen. Like SDE4, RDR2 is partially required for PTGS, which
suggests that there is cross-talk between the RDR6 and RDR2
pathways (A.J. Herr and D.C. Baulcombe, unpublished
work). The RDR2/SDE4 contribution to silencing must be
dependent on a signal from the RDR6 pathway because
silencing is completely lost in the rdr6 mutant. Movement of
a signal from the cytoplasm to the nucleus has precedence in
the observation that cytoplasmically replicating viruses guide
methylation of non-transcribed promoters [58]. The traffic
may not necessarily go both ways. Abundant 24 nt siRNA
directed against a Sine element in Nicotiana benthamiana are
unable to confer resistance to a virus carrying a portion of the
same Sine element [6].
If an immune system can be saturated, improperly maintaining a strong defence against one pathogen may make an
individual plant more susceptible to a different infection. One
way to avoid this problem is to condition amplification of
the defence response to the presence of the pathogen. One
way to avoid this problem is to condition amplification of
the defence response to the presence of the pathogen. In
keeping with this idea, RDR6 amplifies the silencing response
by converting target RNA into a long dsRNA substrate for
DCL; not, it seems, by amplifying dsRNA or siRNAs directly
[70]. The evidence for this comes from experiments that
demonstrate that a dsRNA trigger of silencing corresponding
to only a portion of a target gene, leads to RDR6-dependent
production of secondary siRNAs from the entire length of
the gene – even if the trigger is targeted at the 5 end of the
gene. This does not favour a simple primer-dependent model
of RDR6 activity, because antisense siRNAs from the trigger
can only prime dsRNA synthesis from target RNA sequences
that are 5 of the trigger [70]. Studies in wheat-germ extracts
indicate that ssRNA is readily converted into siRNA even in
the absence of a primer [5]. One possibility is that an initial
siRNA recognition event might license the entire mRNA for
conversion into dsRNA.
An RDR–DCL cycle not only provides a continual supply
of siRNAs for RISC, but also destroys some proportion of
the target RNA. Whether this cycle is robust enough to eliminate the need for endonucleolytic cleavage by RISC is unclear. One potential danger with a robust RDR–DCL cycle is
recruitment of miRNA targets by RDR pathways. To
have miRNA-directed cleavage co-exist harmoniously alongside the defence response, there must be mechanisms to ensure
that transitivity does not run wild on miRNA targets, where
amplification of silencing signal might disrupt the timing or
spatial constraints of regulation. Examination of the siRNA
database and ability to detect miRNA cleavage products
suggest that RDR-dependent transitivity does not take place
on miRNA targets [25,47]. Furthermore, transitivity on
endogene mRNA does not proceed with the same efficiency
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Figure 2 A model for RNA silencing in Arabidopsis
Evidence exists for at least three branches of RNA silencing: one that acts at the chromatin level, one that processes and
effects miRNA regulation and one involved in viral defence and PTGS.
as on transgene mRNA [70], suggesting that there are controls
in place that distinguish self from non-self.
for the generous financial support of the Gatsby Charitable Trust and
a post-doctoral fellowship from Burrough’s Wellcome Fund.
Perspective
The evidence for at least one miRNA pathway and three
different RDR-dependent pathways indicates the diversity of
silencing mechanisms in plants (Figure 2). The observations
that PINHEAD (AGO10) maintains the proper number of
meristem cells [71,72] and ZIPPY (AGO7) ensures proper
timing of adult vegetative structures [73] are reminders that
there may be two additional small RNA-guided effector
complexes that we know little about. In fact, there are
probably others, the activities of six more PPD proteins
remain unaccounted for. The cloning of small RNAs and their
analysis in silencing mutants has been critical for giving shape
to the current silencing landscape in plants. One way forward
may be defining the small-RNA pathways more rigorously
by systematic cloning of small RNAs associated with specific
effector complexes.
Note added in proof
(received 3 September 2004)
Two recent papers provide convincing biochemical evidence
that argonaute proteins are the sequence-specific endonuclease of RISC [74,75]. A third, reporting the crystal structure
of an archaeal argonaute shows that, while the PAZ domain
binds the siRNA, the PIWI domain directs cleavage by an
RNase-H-like mechanism [76].
I thank David Baulcombe and past and present members of the
Baulcombe Laboratory for stimulating discussions. I am also grateful
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Received 23 June 2004
C 2004
Biochemical Society
951