meeting
meeting reportreportDelving into the diversity of silencing pathways
Symposium on MicroRNAs and siRNAs: Biological Functions
and Mechanisms
Silke Dorner, Ana Eulalio, Eric Huntzinger & Elisa Izaurralde+
Max Planck Institute for Developmental Biology, Tübingen, Germany

developments include the elucidation of steps in the biogenesisof repeat-associated small interfering RNAs (rasiRNAs), new ideas
about how siRNAs silence gene expression, how miRNAs repress
translation, and the importance of siRNAs as tools in biologicalresearch and therapeutics.
The discovery of siRNAs, which silence gene expression at
the transcriptional and post-transcriptional level in a sequencedependent manner, has revolutionized the biological sciences.
Since their discovery, it has become apparent that various classesof siRNA exist, which differ in their origin and mode of bio­genesis.
They all associate with proteins of the Argonaute family, whichmediate their functions. Multicellular organisms encode sev­
eral Argonaute paralogues, which fall into two main clades: theArgonaute and the PIWI-like proteins (Carmell et al, 2002). The
elucidation of steps in the biogenetic pathway of small regulatory
RNAs that associate with the PIWI-like proteins was a highlight ofthis meeting.
The Keystone Symposium on MicroRNAs and siRNAs: Biological
Functions and Mechanisms took place between 28 January and2 February 2007, in Keystone, Colorado, USA, and was organized
by D. Bartel and S. Grewal.

Keywords: heterochromatin; miRNAs; silencing; PTGS; RNAi
EMBO reports (2007) 8, 723–729. doi:10.1038/sj.embor.7401015

Introduction
This Keystone Symposium highlighted the diversity of mecha­
nisms by which small interfering RNAs (siRNAs) are generated
and regulate gene expression, and the diverse roles that micro­
RNAs (miRNAs) have in development, cell differentiation and thepathogenesis of human diseases. Since the first meeting of thisseries in 2002, progress in the field has been remarkable. Crucial
Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen,Germany+
Corresponding author. Tel: +49 70 71 601 1350; Fax: +49 70 71 601 1353;
E-mail: elisa.izaurralde@tuebingen.mpg.de
Submitted 20 March 2007; accepted 22 May 2007; published online 29 June 2007

©2007 European Molecular Biology Organization

‘Ping-pong’ mechanism for rasiRNA biogenesis
PIWI-like proteins are required for the establishment and maint­en­ance of the germline. In Drosophila, these proteins are thought
to have a role in silencing retrotransposons and other repetitive
genetic elements, thereby preserving the integrity of the genome.
Last year, several groups reported that small RNAs associatedwith these proteins do not have the characteristic 21-nucleotide
length of miRNAs or siRNAs, but are 25–30 nucleotides in length(reviewed by Parker & Barford, 2006). Furthermore, the biogenesisof these RNAs is independent of Dicer (Vagin et al, 2006), raising
the question of how they are generated.
G. Hannon (Cold Spring Harbor, NY, USA) and M. Siomi(Tokushima, Japan) have provided at least a partial answer to thisquestion (Brennecke et al, 2007; Gunawardane et al, 2007). Bothgroups have analysed small RNAs associated with the Drosophila
PIWI-like protein Argonaute 3 (AGO3). Similarly to the two other
Drosophila PIWI-like proteins—PIWI and Aubergine (AUB)—AGO3
is expressed mainly in the germline, and associates with small 24–27nucleotide RNAs that are complementary to AUB-associated small
RNAs in their first ten nucleotides. The PIWI- and AUB-associatedRNAs match the antisense strand of retrotransposons and repetitive
EMBO reports VOL 8 | NO 8 | 2007 7 2 3

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5’

3’

AGO3

Antisense primary transcript

5’

A

3’

3’ end cleavage

5’

AGO3

3’

5’

A
U

3’

AGO3
3’

5’

A

U

5’

3’
PIWI/AUB

3’

5’

A
U

5’

PIWI/AUB

3’end cleavage

3’
U

5’

3’
PIWI/AUB

Sense primary transcript
3’
A

5’

Fig 1 | ‘Ping-pong’ mechanism for rasiRNA biogenesis. A model showing that the 5' end formation of repeat-associated small interfering RNAs (rasiRNAs)
associated with Argonaute 3 (AGO3) and Aubergine (AUB; or PIWI) are mediated by AUB (or PIWI) and AGO3, respectively. The mechanism by which the 3'
ends of rasiRNAs are generated is still unknown. Scissors represent endonucleolytic cleavage events. This model is based on the studies by Brennecke et al (2007)
and Gunawardane et al (2007).

sequence elements (therefore referred to as rasiRNAs), whereas
AGO3-associated RNAs are derived from the sense strand. PIWI- and
AUB-associated RNAs show a strong bias for uracil (U) at their 5'
ends, whereas AGO3-associated RNAs show a strong preference foradenine (A) at position 10. Together, these observations suggest an
amplification loop mechanism whereby the 5' end of AUB- (or PIWI)associated RNAs are generated by endo­nucleolytic cleavage of pre­cursor transcripts guided by AGO3-associated RNAs. Conversely, the5' ends of AGO3-associated RNAs are generated by endonucleolytic
cleavage guided by rasiRNAs asso­ciated with AUB (or PIWI; Fig 1). In
agreement with this model, Siomi and co-workers have shown thatboth AUB and AGO3 have slicing activity.
Hannon noticed that although 70% of AGO3-, AUB- andPIWI-associated RNAs in Drosophila match retrotransposons andrepetitive sequence elements, they originate from a relatively small
number of loci in the genome. Evidence that two of the genomic
loci from which rasiRNAs are transcribed are indeed importantfor silencing of transposons originates from the observation thatthey overlap with previously characterized master control lociof known transposons. This includes the flamenco locus on the
X chromosome that consists of fragments of transposons—includ­ing gypsy, ZAM and others—which are all oriented in the same
direction and are probably transcribed as a single long transcript.
This transcript could be processed by rasi­RNAs originating fromtranscripts in the reverse orientation.
The observations reported by Hannon and Siomi provide impor­tant insights into the biogenesis of rasiRNAs; nevertheless, manyquestions remain. For example, how are the 3' ends of these RNAs
generated? How and when does this cycle start? Is it nuclear or cyto­
plasmic? Both AUB and AGO3 are predominantly cytoplasmic; bycontrast, PIWI is predominantly nuclear, and its expression pattern
72 4 EMBO reports

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does not overlap fully with that of AGO3, so how are the 5' ends of
PIWI-associated RNAs generated in cells that lack AGO3? Finally,
how are retrotransposons and mobile genetic elements silenced?

MicroRNA-target prediction: how many targets are ‘real’?
MicroRNAs were an important focus of the meeting. There areat least 100 miRNAs in invertebrates and 500 in vertebrates.
Computational predictions of miRNA targets indicate that eachmiRNA might regulate hundreds of different mRNAs. However,
several speakers at this meeting pointed out a potential discrep­
ancy between the number of predicted targets and the observationthat abolishing miRNA activity does not always result in an overt
pheno­type. Indeed, R. Plasterk (Utrecht, the Netherlands) showedthat inactivating individual miRNAs in zebrafish using morpholino
oligo­nucleotides often resulted in no detectable phenotype.
V. Ambros (Hanover, NH, USA) showed that although Drosophila
let-7 has many predicted targets, the main phenotype associated withthe loss of function of this miRNA can be rescued by reducing theexpression levels of a single mRNA target. Flies that lack let-7 havedefective courtship behaviour and do not discriminate between
males and females. However, this complex neurological pheno­
type is restored by reducing the expression of Abrupt, a transcription
factor regulated by let-7, indicating that misregulation of a single,
direct target is responsible for the observed phenotype. Similarly, in
Caenorhabditis elegans, defects associated with the loss of lin-4 andlet-7 miRNAs are suppressed by mutations that reduce the expressionof single targets—that is, lin-14 and lin-41, respectively.
R. Carthew (Evanston, IL, USA) provided an explanation for theseemingly contradictory observations that although miRNAs havehundreds of targets, loss-of-function mutations in miRNA do nothave the expected pleiotropic effects. Studies on the Drosophila©2007 European Molecular Biology Organization

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miR-7 led Carthew and co-workers to propose that miRNAs areincorporated into robust regulatory networks, and that the loss of
one node of the network—for example, the miRNA—has no detect­able consequences when flies are grown under standard laboratory
conditions. Only when the network itself is impaired would theeffects of miRNA loss-of-function be seen (Li & Carthew, 2005).
Another reason why the number of consequential targets for agiven miRNA might be smaller than the number of conserved targets
is that miRNAs might fine-tune target expression, so their absence
results in only minor effects in most circumstances. Finally, miRNAbinding sites might simply be masked by secondary structures orby RNA-binding proteins. R. Schroeder (Vienna, Austria) showedthat the accessibility of an siRNA-binding site is crucial for RNAinduced silencing complex (RISC) activity. W. Filipowicz (Basel,
Switzerland) showed that binding of a protein known as HuR to the
3' untranslated region (UTR) of cationic amino-acid transporter 1(CAT1) mRNA during stress conditions relieves miR-122-mediatedsilencing, probably as a result of the displacement of the miRNA(Bhattacharyya et al, 2006). Therefore, it seems likely that, in addi­
tion to HuR, other RNA-binding proteins are able to counteract
miRNA-mediated silencing. If these proteins were to be expressed
under specific physiological conditions or in a tissue-specific man­ner, then immunity or susceptibility to mi­RNAs could be regulated.
This view was further strengthened by data presented by A. Giraldez(New Haven, CT, USA), who showed that silencing of nanos mRNAby miR-430 occurs in somatic cells but not in the germline, and that
this effect is mediated by 3' UTR sequences outside the miRNAbinding site. Together, these observations suggest that the presenceof a miRNA-binding site is not always predictive of miRNA regula­tion in the cell type under investigation and that the functionality ofmiRNA-binding sites should be experimentally validated.
Although, at present, it is not possible to predict the accessibil­
ity of miRNA-binding sites in vivo, the increasing number of experi­mentally validated miRNA-binding sites is improving the accuracyof target predictions. D. Bartel (Cambridge, MA, USA) and C. Burge(Cambridge, MA, USA) observed that conserved seed matches are
more strongly repressed and are more frequently associated withadditional determinants. Bartel found that the position of the miRNAbinding site within the 3' UTR has an important role: sites that areclose to the open reading frame or to the 3' end of the transcript are
more likely to be regulated than sites more centrally located within
3' UTRs, and sites that are near to each other tend to act coop­eratively. Sequence context also influences the likelihood that asite will be regulated in vivo: sites that are embedded in A+U-richsequences are more likely to be regulated. Burge observed that an A
or U at position 9 of the target site is over-represented among ‘good’
miRNA-binding sites regardless of complementarity. The rules thatare becoming apparent from these talks are based on the analysis ofa large number of human miRNA targets. It would be interesting toknow whether they hold true in other organisms such as Drosophila
in which most 3' UTRs are A+U-rich.

Role of microRNAs in cell differentiation
Several talks addressed the biological role of miRNAs using cellularand developmental genetic approaches in both tissue culture andin living organisms. Some miRNAs have a tissue-specific pattern ofexpression and this provides a first hint on their potential biologi­
cal function. P. Sharp (Cambridge, MA, USA) discussed how miRNA
expression changes during the matur­ation of T lymphocytes. These©2007 European Molecular Biology Organization

miR-375 knockdown

Wild-typeFig 2 | Role of miR-375 in pancreatic islet cell organization. Knockdown of
miR-375 using a morpholino oligonucleotide complementary to the miR-375
precursor causes abnormalities (arrows) in pancreatic islet organization inzebrafish. Image courtesy of R. Plasterk and R. Ketting (Utrecht,
the Netherlands).

changes can be correlated with stage-specific cell processes, sug­gesting that miRNAs have important roles in cell differentiation(Neilson et al, 2007). In particular, miR-181 was found to be upregu­
lated in double-positive stage thymocytes. These results are in agree­
ment with the role of miR-181 in T-cell development as reported by
C.Z. Chen (Standford, CA, USA; Chen et al, 2004).
Other ways to elucidate miRNA function is by direct knockoutof miRNA genes or by expressing oligonucleotides that interferewith their function. J. Krutzfeldt (of M. Stoffel’s group, Zürich,
Switzerland) reported a crucial role for miR-375 in the functionof pancreatic islet cells, which secret insulin. For example, new­born mice that lack miR-375 seem normal, but the adults sufferfrom hyperglycaemia owing to a decreased pancreatic β-cell mass
and an increased number of glucagon-secreting α-cells. The role ofmiR-375 in the development of pancreatic islet cells is conserved,
as a similar aberrant pancreatic islet morphology was observedby Plasterk in zebrafish embryos injected with morpholino
oligonucleo­tides complementary to the miR-375 precursor (Fig 2).
Three further studies presented at the meeting illustrate the roleof miRNAs in cell differentiation. Sharp showed that knockout of themurine miR-290–295 cluster leads to embryonic lethality, althoughsome ‘escapers’ grow to adulthood. Female escapers have no ovaries,
indicating an essential role for this cluster in the specification and/or maintenance of the female germline. K. Ivey (San Francisco, CA,
USA) found miR-1 and miR-133 to have a role in the development
EMBO reports VOL 8 | NO 8 | 2007 7 2 5reviewsof mesodermal precursors, and A. Dutta (Charlottesville, VA, USA)
showed that miR-206 was required for muscle-cell differentiation in
response to serum starvation (Kim et al, 2006).

MicroRNAs in human disease
C. Croce (Columbus, OH, USA) emphasized the emerging evidence
that miRNAs have important roles in the pathogenesis of several
human diseases, including cancers and metabolic disorders. Indeed,
many miRNA genes are located in chromosomal regions that arealtered in cancer patients. The expression of some miRNAs is reduced
in some cancer cells, suggesting that these miRNAs downregulate
oncogenes. Croce presented evidence indicating that miR-15 andmiR-16 act as tumour suppressors in 68% of chronic leukaemias, andthat an important target of these miRNAs is B-cell lymphoma 2 (Bcl2;
Cimmino et al, 2005). A. Krichevsky (Boston, MA, USA) reported thatmiR-21 expression is high in solid tumours, and inhibitors of miR-21
prevent tumour growth (Chan et al, 2005). Bartel showed that theloss of let-7-directed repression of the high mobility group protein
HMG2A oncogene provides a mechanism for transformation (Mayret al, 2007). Therefore, miRNAs regulate various aspects of tumour
growth and might represent a new class of therapeutic targets. In
addition, changes in miRNA expression profiles correlate with thedifferentiation state of the tumours, as shown by Croce, suggestingthat miRNA profiles could be used for cancer diagnosis.

Mechanisms of microRNA-mediated gene silencing
Despite the remarkable progress we have seen in understanding
miRNA function and target prediction, the mechanisms by which
miRNAs regulate gene expression remain controversial. Initially,animal miRNAs were reported to repress translation without sig­
nificantly affecting mRNA levels. More recently, several reports
have shown that animal miRNAs can induce significant degrada­tion of target mRNAs (reviewed by Pillai et al, 2006). Consistently,
trans­cripts that are upregulated in cells in which the miRNA path­way is inhibited, for example, by depletion of Dicer or Argonaute
proteins, are enriched in predicted and validated miRNA targets.
Conversely, ectopic expression of specific miRNAs in cells inwhich they are not normally present leads to a reduction of the lev­els of transcripts containing binding sites for the miRNA (reviewed
by Pillai et al, 2006).
Sharp reported that during the maturation of T lymphocytes,
upregu­lation of a given miRNA correlates with the downregulation oftrans­cripts that contain complementary binding sites, again suggest­
ing that mRNA levels are regulated. However, changes in protein andmRNA levels are not always correlated; some targets are regulated
mainly at the protein level without detectable changes in the amountof trans­cript present, whereas others are regulated mainly at the mRNAlevel. Sharp found that whether miRNAs elicit decay or translational
repression depends on the structure of the miRNA–target duplexes(Alemán et al, 2007). With a few known exceptions—such as whenthe miRNA is fully complementary to the target—mRNA decay by
miRNAs in animal cells does not occur through endonucleolytic
cleavage by the Argonaute proteins, but rather by directing mRNAs to
the general mRNA degradation machinery, therefore accelerating theirdecay (Behm-Ansmant et al, 2006; reviewed by Pillai et al, 2006).
Although it seems that we are agreed that miRNAs trigger degra­
dation of their targets to differing extents, two questions remain. Is
mRNA decay a consequence of translational repression? And, more
importantly, how is translation regulated? Recent studies have shown72 6 EMBO reports

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me e t ing rep or tthat miRNAs inhibit the initiation of translation, as mRNAs trans­
lated through cap-independent mechanisms were shown to escapemiRNA-mediated silencing. Other studies have suggested that trans­
lation inhibition occurs after initiation, based on the observationthat miRNAs and some targets remain associated with polysomes.
Furthermore, it has been shown that mRNAs translated through capindependent mechanisms are subject to miRNA regulation and that
regulation occurs by a ribosomal drop-off mechanism (reviewed by
Pillai et al, 2006).
Two reports at this meeting added a new turn to this already com­plex picture. M. Kiriakidou (Philadelphia, PA, USA) presented theunexpected observation that the central domain of Argonaute pro­teins has sequence similarities with the cytoplasmic trans­lation ini­
tiation factor eIF4E. This protein binds to the cap structure of mRNAs
by stacking the methylated base of the cap between two tryptophans.
At the equivalent position of the tryptophans in eIF4E, Argonaute
proteins have phenylalanines, which could mediate a similar inter­action. Consistently, Kiriakidou showed that human AGO2 binds
m7GTP-sepharose beads and that cap analogues—but not GTP—
compete with this binding. Human AGO2 silences the expressionof a luciferase reporter when it is artificially tethered to the 3' UTRof the reporter mRNA. When the two phenylalanines are changed tovalines, this repression is abolished. Conversely, mutations of phe­nylalanine to tryptophan, which are expected to increase the affinityof the protein for the cap structure, increased the silencing activity.
Kiriakidou’s results therefore provide support for a mechanism bywhich translation is inhibited at the initiation step by displacementof eIF4E from the cap structure.
R. Shiekhattar (Barcelona, Spain) showed that eIF6 and large
ribosomal subunits associate with AGO2 in human cells. eIF6
interacts with large ribosomal subunits and prevents their prema­ture association with the small subunit (Ceci et al, 2003). Therefore,
recruitment of eIF6 by RISC might repress translation at the initia­
tion and/or elongation step by preventing large subunit joining.
An explanation for these different models could be that miRNAs
regulate gene expression by different mechanisms, although thepossibility that some of the discrepancies are due to differences inexperimental approaches cannot be excluded.
In contrast to most animal miRNAs, plant miRNAs in general
exhibit a more extensive complementarity to their targets and arethought to silence gene expression by promoting endonucleolytic
cleavage of their targets in a similar way to siRNAs. O. Voinnet(Strasbourg, France) convinced us that the mode of action of plant
miRNAs also includes an important contribution of translational
repression to silencing. In a genetic screen designed to isolate
genes required for miRNA biogenesis and function in Arabidopsis,
Voinnet identified mutations that affect miRNA biogenesis, as well
as some that affect endonucleolytic cleavage of the mRNA target
without affecting miRNA levels. Another class of mutations restored
protein expression levels from the reporter, although endonucleo­
lytic mRNA cleavage was still occurring. This indicates that thesemutants affect translational silencing, but not mRNA decay by
miRNAs. Further analysis of these mutants will certainly provide
new insights into how miRNAs silence gene expression.

New regulatory RNAs
The advent of high-throughput and ‘454’ sequencing techniques hasboosted the field of small regulatory RNAs. The power of these tech­
nologies is particularly impressive when applied to the analysis of©2007 European Molecular Biology Organization

me e t i n g rep or tpools of small RNAs present in wild-type backgrounds or in knock­outs of components of silencing pathways. This allows the detectionof new regulatory RNAs and also provides important informationon their biogenesis. For example, 46 new miRNA hairpins weredetected by comparing wild-type and Dicer-knockout embryonicstem cells (Sharp). D. Baulcombe (Norwich, UK) discussed resultsfrom a high-throughput sequencing of siRNAs and miRNAs using
wild-type and mutant Arabidopsis plants. He showed that 90% of
plant siRNAs are generated by a plant-specific polymerase—pol
IVa+b—and are derived from transposons and retrotransposons.
In-depth sequencing analysis also revealed a network of siRNAs in
which primary siRNAs lead to the generation of secondary siRNAs.
In addition, Baulcombe and co-workers identified miRNAs andmiRNA-like RNAs in Chlamydomonas reinhardii. Interestingly, theyfound phased small RNAs in 21-nucelotide increments relative tothe start cleavage site, although no recognizable RNA-dependent
RNA polymerase has been identified in this organism. J. Carrington(Covallis, OR, USA) reported the identification of 39 new miRNA
families in Arabidopsis that are not conserved in rice, indicating thatthese represent new mi­RNAs (Fahlgren et al, 2007).
C. Mello (Worcester, MA, USA) presented results from 454
sequencing of a new class of small RNAs in C. elegans that are
dependent on the Dicer-related helicase DRH-3. Out of 48,000
RNA molecules, 11,000 were small 22-mers. Of these, 98% started
with guanine, approximately 50% hit annotated genes (about 97%
antisense and 3% sense) and 50% corresponded to noncoding andrepetitive sequences. Therefore, Mello speculated that a significant
fraction of the genome is regulated by the silencing machinery.
T. Tuschl (New York, NY, USA) reported sequencing 250 smallRNA libraries from 26 different human organs. 65% of clones inthese libraries represent miRNAs. It is estimated that there are 500
miRNA genes in humans, and about 39 of these are not found inany other organism. Carthew reported 101 new miRNAs that areconserved in all 12 Drosophila species, and about 212 that are notconserved and are expressed at low levels.
Some general rules can be derived from these genomic studies:ubiquitous miRNAs are generally expressed at high levels, whereas
species-specific miRNAs are expressed at low levels, and for some ofthese, no targets have been identified. The theme that species-specificmiRNAs are generally expressed at low levels, belong to single-genefamilies and have no detectable targets was reiterated several times
during the meeting (Carthew and Carrington). N. Rajewsky (Berlin,
Germany) proposed that most weakly expressed miRNAs—in partic­
ular, lineage-specific miRNAs—have no biological function, and areon an evolutionary path either to generate a new function or to beeliminated. The observation that new miRNAs are expressed at low
levels also explains why their acquisition is tolerated, and this in turn
favours their evolution (Chen & Rajewsky, 2007).

Diversity of silencing pathways
Silencing pathways have evolved to perform diverse biological
functions, some of which seem to be organism- or species-specific.
M. Gorovsky (Rochester, NY, USA) reported progress on the eluci­
dation of how the RNA-silencing machinery is involved in germline
DNA elimination in Tetrahymena thermophila. T. thermo­phila has
two nuclei in each cell: a transcriptionally active, polyploid macro­
nucleus, and a transcriptionally silent diploid germline micro­
nucleus. During sexual division, the old macronucleus is destroyed
and a new macronucleus is formed from the micro­nucleus. As part©2007 European Molecular Biology Organizationreviewsof this process, DNA sequences known as internal eliminatedsequences (IESs), which represent 10–15% of the germline DNA,are eliminated. IES elimination requires complementary 28-nucleo­
tide small regulatory RNAs (scan RNAs or scnRNAs), an Argonaute
protein (Twi1), a Dicer-like protein DCL1, a DExH-box RNA
helicase and additional proteins, some of which have similarities
with histone-modification and chromodomain proteins. Gorovsky
also identified two other proteins required for this process. These
proteins are characterized by the presence of glycine–tryptophan
repeats and are therefore reminiscent of GW182 (glycine–
tryptophan protein of 182 KDa) and TNRCA–C (trinucleotiderepeat-containing proteins A–C), which interact with the Argonaute
proteins in C. elegans, Drosophila and humans (Behm-Ansmant et al,2006 and references therein).
In C. elegans, the Argonaute protein family has expanded withmore than 23 Argonaute paralogues, some of which have roles inprimary silencing events and others in transitive RNA interference(RNAi; Yigit et al, 2006). Transitive RNAi is the phenomenon bywhich a small number of RNA molecules can silence a gene overmore than 80 animal generations, suggesting an amplification of
the primary RNA trigger and the generation of secondary siRNAs.
Amplification is mediated by an RNA-dependent RNA polymer­
ase but the precise mechanism by which this enzyme operates hasremained elusive. Originally it was thought that the primary siRNA
acted as a primer to direct the synthesis of a strand complemen­tary to the target. A. Fire (Stanford, CA, USA) and Plasterk showedthat this is not the case (Pak & Fire, 2007; Sijen et al, 2007). Bothgroups have characterized secondary siRNAs and have shown thatthey carry 5' di- or triphosphates, are of antisense polarity and starta few nucleotides upstream of the primary siRNA. On the basisof these observations, they proposed that each secondary siRNAderives from an independent, unprimed RNA synthesis event by
the RNA-dependent RNA polymerase. In the current model, pri­mary siRNAs guide the recruitment of this polymerase to target
mRNAs. This leads to the de novo, unprimed synthesis of antisense
RNAs, which are incorporated into silencing complexes by an
unknown mechanism.
Finally, silencing pathways are highly elaborate in plants. The
Arabidopsis genome encodes six RNA-dependent RNA polymer­
ases, four Dicer-like proteins and 10 Argonautes. Baulcombe
proposed that these proteins form functional modules, which
combine to produce different silencing pathways that might act at
the trans­criptional or post-transcriptional levels.
So far, Drosophila was thought to be exceptionally simple with
only two Argonaute-like proteins: AGO1 having a role in the miRNA
pathway, and AGO2 acting in RNAi. P. Zamore (Wocester, MA,
USA) reported that silencing pathways in Drosophila are not as spe­cific as previously thought and that miR-277 is loaded into AGO2containing RISC, although it is processed by Drosha and Dicer-1.
However, to be loaded into AGO2-containing RISC, Dicer-2 andR2D2 are required. Further analysis showed that it is the structureof the duplex, rather than the biogenetic pathway, dictating withwhich AGO protein an RNA duplex associates. For example, a cen­tral mismatch in the pre-miRNA disfavours Dicer-2–R2D2 bind­ing and hence AGO2 loading, whereas fully complementary RNA
duplexes associating with AGO1 will unwind very slowly. Clearly,
the challenge now is to determine which endogenous miRNAs
associate with AGO2 and which endo­genous targets are regulatedby endonucleolytic cleavage.
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Spreading
SHREC

SHREC

HP

HP

Repeat elements

DCR1

HP
Pol II

H3K9meClr4 complex
RDRC RITS
siRNAsFig 3 | Role of silencing in heterochromatin formation. The RNA interferencemachinery, consisting of the RNA-induced initiation of transcriptional genesilencing (RITS) complex, Dicer-1 (DCR1) and the RNA-directed RNA
polymerase complex (RDRC), processes transcripts derived from repeat
elements into small interfering RNAs (siRNAs). These siRNAs might silencegene expression post-transcriptionally in cis. The Clr4 complex methylates
histone 3 lysine 9 (H3K9me), which in turn facilitates stable tethering of thesilencing machinery to chromatin through RITS and also allows recruitmentof heterochromatin-binding proteins (HPs), such as Swi6/HP1. HP binding
creates a platform that allows the spread of Snf2/Hdac-containing repressor
complex (SHREC) across an entire domain. SHREC can also be recruited
to some genomic loci through specific DNA-binding proteins in a process
independent of the silencing machinery (not shown). This model is based on
the study by Sugiyama et al, 2007.

S. Elgin (St Louis, MO, USA) reported that Drosophila HP1a, a
protein that localizes to heterochromatin, interacts with the aminoterminal domain of PIWI but not with AGO1–3 or AUB, as shownby a yeast two-hybrid study performed in collaboration with H. Lin(New Haven, CN, USA). HP1 dimerization is required for this inter­action. Immunofluorescent staining of polytene chromosomes
revealed the presence of PIWI in both euchromatic arms andhetero­chromatic regions. The heterochromatic staining is sensitive
to RNaseH treatment, suggesting that PIWI-associated RNAs might
have a role in targeting PIWI to heterochromatic regions through theformation of RNA–DNA hybrids.
In contrast to animals, transcriptional gene silencing inplants involves DNA methylation. De novo DNA methylation in
Arabidopsis thaliana requires components of the silencing machin­
ery including AGO4, Dicer-like-3 (DCL3), an RNA-dependent RNA
polymerase 2 (RDR2), and the plant-specific polymerase (pol IVb).
M. Matzke (Vienna, Austria) reported the characterization of com­ponents of the plant-specific RNA polymerase polIVb. Endogenous
targets in Arabidopsis were identified and these are mainly in inter­genic regions and plant genes located primarily in euchromatin.
Matzke proposed a basal inactivation mechanism through thispathway, which can either be reversed to euchromatin or reinforced
in heterochromatin.

Conclusions
Small regulatory RNAs are at the forefront of biomedical research.
However, many questions remain about their biogenesis, biological
function and mechanisms of action. There is no doubt that answers
to many of these remaining questions in this fast-moving anddynamic field will be provided in the next meeting of this series, anevent to which we can all look forward.

Transcriptional gene silencing

Acknowledgements

Most of the information we have on how silencing pathways regu­
late gene expression at the transcriptional level derives from
Schizosaccharomyces pombe and plants. S. Grewal (Bethesda,
MD, USA) previously reported the characterization of the RNAinduced initiation of transcriptional gene silencing (RITS) complex
in S. pombe, which consists of the Argonaute protein AGO1,
Tas3 and Chip1. Grewal showed that RITS associates with hetero­chrom­atic regions, and spreads in cis in a process requiring
heterochrom­atin-binding proteins (HPs), such as Swi6/HP1 (Fig 3).
The S. pombe genome also encodes an RNA-directed RNA
polymerase RDRC (containing Rdrp1) that, together with compo­
nents of the RITS complex, localizes in heterochromatic regionsof the genome, which are also marked by methylation of histone
H3 at lysine 9 (H3K9me) catalysed by the Clr4 complex (Fig 3).
Grewal and co-workers have proposed that the RNA-silencing
machinery processes transcripts derived from these loci into siR­
NAs, which might neutralize future invasion by similar sequences.
Grewal also described the multi-enzyme effector complex SHREC(Snf2/Hdac-containing repressor complex). This heterotetramericcomplex—consisting of Clr1, Clr2, Clr3 and Mit1—mediates hetero­chromatic transcriptional gene silencing in S. pombe by assisting
the assembly of higher-order chromatin structures that are crucial
for hetero­chromatin function (Sugiyama et al, 2007). SHREC isrecruited to various genomic locations through mechanisms
involving the RNA silencing machinery and/or sequence-specificDNA-binding proteins (Fig 3).

We are grateful to D. Bartel and S. Grewal for organizing this highly
stimulating meeting and to the speakers whom we consulted about the
text. We apologize to the many participants whose excellent contributions
could not be mentioned owing to space constraints. We thank D. Thomas
for helpful comments on the report. S.D. and E.I. are supported by the
Max Planck Society; A.E. and E.H. are recipients of fellowships from thePortuguese Foundation for Science and Technology and the European
Molecular Biology Organization, respectively.

72 8 EMBO reports

VOL 8 | NO 8 | 2007

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From left to right: Silke Dorner, Elisa Izaurralde, Eric Huntzinger& Ana Eulalio

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