MEETING REVIEW 1635

The regulation of genes andgenomes by small RNAs
Victor Ambros1,* and Xuemei Chen2
A recent Keystone Symposium on ‘MicroRNAs and siRNAs:
Biological Functions and Mechanisms’ was organized by David
Bartel and Shiv Grewal (and was held in conjunction with ‘RNAi
for Target Validation and as a Therapeutic’, organized by
Stephen Friend and John Maraganore). The ‘MicroRNAs andsiRNAs’ meeting brought together scientists working on diverse
biological aspects of small regulatory RNAs, including
microRNAs, small interfering RNAs (siRNAs) and Piwi-interacting
RNAs (piRNAs and rasiRNAs). Among the themes discussed were
the diversity of small regulatory RNAs and their developmental
functions, their biogenesis, the identification of their regulatory
targets, their mechanisms of action, and their roles in theelaboration of multicellular complexity.

Introduction
MicroRNAs, small interfering RNAs (siRNAs) and Piwi-interacting
RNAs (piRNAs) are small regulatory RNAs that are unified by theirassociation with Argonaute-family proteins and by their functions
as regulatory RNAs that direct the binding of protein complexes tospecific nucleic acid sequences. These small RNAs can exert
regulation at the transcriptional level, by affecting chromatin
structure, or post-transcriptionally, by affecting mRNA stability ortranslation.
Developmental functions of microRNAs
Animals and plants have hundreds of distinct microRNA genes,
whose regulatory roles in development have been implicated bytheir diverse expression patterns and, in some cases, confirmed bygenetic studies in model organisms and in humans (Kloosterman and
Plasterk, 2006; Jones-Rhoades et al., 2006). Of the three mainclasses of small regulatory RNAs discussed at the meeting –microRNAs, siRNAs and piRNAs (Fig. 1) – the developmental roles
for microRNAs are the most-clearly evident.
Richard Carthew (Northwestern University, Evanston, IL, USA)described how miR-7 functions in an intricate network of Notch andepidermal growth factor receptor (EGFR) signaling pathways tospecify photoreceptors (Li and Carthew, 2005) and chordotonal
sensory organ precursors in Drosophila. Carthew proposed thatmicroRNAs such as miR-7 function in such networks to confer
robustness on developmental decisions and to provide genetic
buffering under adverse physiological conditions. Indeed, Carthew
reported that exposing developing mir-7 mutant fly eyes totemperature cycles disrupts regular ommatidial patterning. Anotherexample of a microRNA-modulated signaling pathway in
Drosophila was discussed by Eric Lai (Sloan-Kettering Institute,
New York, NY, USA). miR-315 was identified in a cell-based screen
as a potentiator of Wingless (Wg) signaling. Consistent with a role1

Dartmouth Medical School, Hanover, NH 03755, USA. 2UC Riverside, Riverside, CA
92521, USA.
*Author for correspondence (e-mail: vambros@dartmouth.edu)

for miR-315 in promoting Wg signaling in vivo, among the targetsof miR-315 are the negative regulators of the Wg pathway, Axin andNotum. Moreover, the overexpression of miR-315 in thepresumptive thorax of developing larvae caused dramaticdevelopmental transformations associated with excess Wg activity.
MicroRNAs play important roles in the developmental
progression of certain cell lineages and fates. Interestingly, despite
the deep phylogenic conservation of some microRNA genes(Pasquinelli et al., 2000), a conserved microRNA can regulatedifferent genetic pathways and developmental processes in different
organisms. The genetic analysis of Drosophila let-7 by Nick Sokol
of Victor Ambros’s laboratory (Dartmouth Medical School,
Hanover, NH, USA) revealed that, unlike the worm let-7, which
controls developmental transitions mainly in hypodermal cells(Reinhart et al., 2000), the main function of fly let-7 seems to be tocontrol events in the development of the adult nervous system. In therespective worm and fly let-7 pathways, the key direct targets of
let-7 also differ.
Phillip Sharp (Massachusetts Institute of Technology, Cambridge,
MA, USA) compared the expression profiles of microRNAs andtheir computationally predicted targets during T cell development,
and identified at least one microRNA for each stage whose
expression inversely correlated with its predicted-target levels. Of
particular note was miR-181 (also known as Mirn181a-2 – Mouse
Genome Informatics), which had previously been shown to play arole in mouse lymphocyte cell fate specification (Chen et al., 2004),
and was found by Sharp to be enriched in double-positive (DP) cells(Neilson et al., 2007). These observations indicate that miR-181 has
a role in the positive selection of DP lymphocytes in response to
antigen presentation. Chang-Zheng Chen (Stanford University, Palo
Alto, CA, USA) reported how the manipulation of miR-181 levels
in T cells modulates their sensitivity to T cell-receptor signaling, andhence their response to antigens, indicating that miR-181 may act asa ‘rheostat’ to tune T cell sensitivity at various stages of T cell
development. Chen pointed out that this role of miR-181 inlymphocyte development exemplifies how microRNAs may beparticularly suited to the control of delicately regulated
developmental pathways.
Nikolaus Rajewsky (Max-Delbruck-Centrum, Berlin, Germany)
presented results of a collaborative project from his and Timothy
Bender’s laboratory (Harvard Medical School, Boston, MA, USA)showing that, in mouse, miR-150 (also known as Mirn150 – Mouse
Genome Informatics) plays a role in lymphocyte maturation,
possibly via the regulation of the transcription factor cMyb, a directtarget of miR-150. In transgenic mouse models, moderate ectopic
expression of miR-150 or a graded reduction of cMyb blocks thedevelopment of mature lymphocytes, suggesting that lymphocyte
maturation is sensitive to the dosage of miR-150, which may
modulate cMyb levels at crucial times.
Jan Krützfeldt (from Markus Stoffel’s group, Institute of
Molecular Systems Biology, Zurich, Switzerland) described mir-375(also known as Mirn375 – Mouse Genome Informatics)-knockout
mouse phenotypes, in which pancreatic islets display abnormalarchitecture and a disruption in the balance between alpha and beta
cells. Previous overexpression experiments with miR-375 had
indicated that it acts in the regulation of insulin secretion by islet
cells (Poy et al., 2004). These new findings suggest an additionalrole for miR-375 in pancreatic development. Similar findings werereported by Ronald Plasterk (Hubrecht Laboratory, Utrecht,

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Development 134, 1635-1641 (2007) doi:10.1242/dev.002006

1636 MEETING REVIEWADevelopment 134 (9)B

MIR genesAGO

Pol II

Heterochromatin
maintenance

Pol II Pol IV (?) in plants

C
Multipletransposons

Pol II?

Nascenttranscript

miRNA
precursors
DROSHA
DICER

AGO-boundmiRNA

Homologoustransposon

Aub

RdRP
siRNA
precursor

AGO3
Piwi

AGO

AGO

AGO3

Aub
Aub

Dicer

AGO

Piwi
AGO3

Aub

AGO-bound
AGO
miRNA

AGO

Piwi

Key
Developmental
process 1

Developmental
process 2

Repeated DNA
H3-K9 methylation

Key
Strong contribution
Weak contribution

Netherlands) for the zebrafish miR-375. Knockdown of miR-375
with morpholino oligonucleotides directed against either the mature
microRNA or the stem of the miR-375 precursor caused similar
abnormalities in pancreatic islet organization, strongly supporting
the specificity of this phenotype.
Cell type-specific or organ-specific microRNAs are generally
considered to be potential determinants of organ or cell type identity(Plasterk, 2006). What role do microRNAs play in pluripotent
embryonic stem (ES) cells? Sharp reported the initialcharacterization of a knockout mouse that lacks mir-290 through to
mir-295 (mir-290–295), an ES cell-specific microRNA cluster(Houbaviy et al., 2003). Homozygous mir-290–295–/– animals often
die as embryos, although some grow to adulthood. Significantly,
female survivors are infertile due to an absence of germ cells,
indicating that these microRNAs may function in the maintenanceof pluripotent cells in embryos and in the female germ line.
miR-133 and miR-1 are expressed in mouse mesodermal lineages,
and at particularly high levels in muscle. This muscle-specificexpression is evolutionarily conserved among animals (Nguyen and
Frasch, 2006). Kathryn Ivey (from Deepak Srivastava’s group,
Gladstone Institutes and University of California, San Francisco,
CA, USA) used lentivirus vectors to overexpress miR-1 and miR133 during the in vitro development of embryoid bodies from mouse
ES cells, and showed that distinct steps in muscle development are
specified by cooperative, followed by opposing, interactions
between miR-1 and miR-133. These findings provide insight into

other tissue- or organ-specific microRNAs, where their functionalroles may be dynamic, changing and complex. A previously
published characterization of Drosophila mir-1-knockout mutants(Sokol and Ambros, 2005; Kwon et al., 2005) suggested that miR-1
maintains differentiated muscle during larval growth. In mice,knockout of one of the two mir-1 loci (mir-1-2, also known as
Mirn1-2), as reported by Srivastava’s laboratory, leads to heart
defects, including apparent hyperplasia of myocytes. So, although
the mir-1 sequence and its muscle-specific expression is essentially
identical between insects and mammals, it is not yet clear whether
miR-1 function is entirely conserved. Anindya Dutta (University ofVirginia, Charlottesville, VA, USA) reported a function for miR-206
in the myogenic differentiation of mouse C2C12 cells – a model forregenerative myogenesis – in culture (Buckingham, 2006). When
C2C12 cells are removed from serum, they produce differentiatedmuscle and show miR-1, miR-133 and miR-206 upregulation.
miR-206 appears to be required for muscle differentiation in
response to serum starvation.
microRNAs and cancer
Carlo Croce (Ohio State University College of Medicine, Columbus,
OH, USA) discussed the importance of reliable diagnostic andprognostic markers for cancer, and how microRNA expression
patterns appear to be more reliable than mRNA expression patterns
for distinguishing the tissues of origin of human tumors (Lu et al.,2005). Croce reported that both MIR-15 and MIR-16 are tumor

DEVELOPMENT

Fig. 1. The biogenesis and function of endogenous small RNAs in animals, and in plants and fungi. (A) In animals, microRNA (MIR) genes(top) are transcribed by Pol II into microRNA (miRNA) precursors, which are processed by DROSHA and DICER (DICER1) into miRNAs. The miRNAs
are bound by an effector protein Argonaute (AGO) and cause the cleavage or translational inhibition of target mRNAs. In animals, despite the factthat each miRNA is able to regulate scores of target transcripts, genetic studies show that one major target underlies the role of a miRNA in adevelopmental process. Related miRNAs (light and dark blue) often have shared predicted targets, but genetic studies reveal that members of amiRNA family may have distinct developmental functions. (B) Endogenous siRNAs in S. pombe and in plants are generated from long transcripts
from repeated DNA or transposons via the activities of RNA-dependent RNA polymerases (RdRP) and Dicer. Histone H3 lysine 9 (H3-K9) methylationand the siRNAs complexed with AGO act in a self-reinforcing loop to maintain heterochromatin at the loci. (C) Via an unknown mechanism, repeatassociated siRNAs (rasiRNAs) in Drosophila are produced from a master locus composed of multiple transposons. These small RNAs bind the Piwi
subfamily of Argonaute proteins – Piwi, Aubergine (Aub) and AGO3 – and target homologous transposons scattered around the genome forsilencing. An existing rasiRNA guides the cleavage of a target RNA through the slicer activity of the associated Piwi subfamily of Argonaute protein
to lead to the production of a second rasiRNA.

suppressors in chronic lymphocytic leukemia (CLL), and that theytarget B-cell CLL/lymphoma 2 (BCL2). microRNA expression
patterns distinguish an aggressive from an indolent form of CLL,
potentially permitting earlier intervention with appropriate therapy.
Similarly, measurements of MIR-155 and LET-7 levels distinguishStage-1 human lung cancers of poor prognosis from those with abetter prognosis far sooner than alternate assays.
Anna Krichevsky (Brigham and Women’s Hospital and Harvard
Medical School, Boston, MA, USA), in collaboration with Khalid
Shah (Massachusetts General Hospital, Boston, MA, USA),revealed a role for MIR-21 (also known as MIRN21 – Human Gene
Nomenclature Database) in mammalian brain tumors. The pretreatment of human brain tumor cells with anti-MIR-21
oligonucleotides inhibits their tumorigenicity when transplanted intomice. MIR-21 overexpression is a hallmark of certain classes ofbrain tumors, so these results suggest that MIR-21 could indeed
contribute to the malignancy of these tumor cells, probably via therepression of one or more tumor suppressor genes.
The converse situation, where tumor-suppressive microRNAs
may function to repress oncogenes, was illustrated by David Bartel(Whitehead Institute and MIT, Cambridge, MA, USA). The
oncogenic behavior of certain mutated forms of High mobility group
A2 (HMGA2) in humans is induced by the deletion of the HMGA2
3⬘ UTR (Mayr et al., 2007), which contains several evolutionarily
conserved sites for LET-7, a microRNA with potential tumorsuppressive activity (Johnson et al., 2005).
Finding and validating microRNA targets inanimals
Current strategies to identify the regulatory targets of microRNAs
include the computational prediction of base-pair matches (either
near-perfect, as in plants, or partial base-pairing, as is more common
in animals), followed by experimental validation. A central principleof animal microRNA target identification is the occurrence ofcomplementary sequences in 3⬘ untranslated regions (UTRs), andmatches to the ‘seed’ region (nucleotides 1-8, from the 5⬘ end) of the
microRNA. These relatively lax criteria can result in numerous
targets being predicted for a given microRNA. However, many
predicted interactions are evolutionarily conserved, indicating thatmany animal genes are under selective pressure to maintainmicroRNA target sites.
Interestingly, as Carthew discussed, experimental evidence
suggests a greater functional specificity for microRNA-targeting
than is often computationally predicted. Ambros elaborated thispoint in reviewing the role of let-7 and lin-4 in worm developmental
timing. Although lin-4 and let-7 are each predicted to have many
conserved targets (Lall et al., 2006), the pleiotropic phenotypes that
result from the loss of either microRNA can be suppressed by themutation of just one particular target gene (Ambros, 1989; Slack etal., 2000). Ambros reported that this also applies to let-7 function in
Drosophila, where the severe behavioral defects of the let-7 deletionmutant are efficiently suppressed by the mutation of a single
predicted target, the abrupt transcription factor. These results
suggest that the effects of microRNAs on gene networks may not be
immediately apparent from computational predictions alone, andthat genetic analysis may be the most definitive way to determine
the relative phenotypic impact of specific predicted interactions.
Nevertheless, computational methods can efficiently identify
candidate targets, and improvements in these methodologies were
presented by Chris Burge (Massachusetts Institute of Technology,
Cambridge, MA, USA) and Bartel. From separate studies, a refined
approach to seed-match target prediction was presented. Both

MEETING REVIEW 1637

groups analyzed data from experiments in which microRNA levels
were perturbed in various ways. The resultant changes in target
mRNA levels were monitored by microarray hybridization analysis.
The strength of a microRNA-target interaction, as revealed by thelevel of target mRNA knockdown, reveals how features of the target
site (of a particular microRNA) can influence these interactions.
Bartel reported that favorable target-site features include the lengthand position within the microRNA of the seed match, A/U-richsequences immediately surrounding the seed match, and the distanceof the site from the middle of the UTR. Curiously, Burge found that
an A or U at position 9 of the target site was an efficient predictor ofan effective interaction, regardless of the base-pairing at that
position. Burge also reported that endogenous vertebrate
microRNA-targeting follows similar or identical rules to that ofexogenous microRNAs. Both Burge and Bartel found that conserved
seed matches tend to provide more efficient knockdown than nonconserved seed matches, perhaps in part due to the evolutionarily
conserved favorable features, such as those reported above. They
also found that different types of seed matches with specific patternsof complementarity to mRNAs consistently mediate different levels
of mRNA downregulation.
Ultimately, assaying only the changes in target mRNAs levels(without also assaying the corresponding proteins) is not sufficient
to characterize all functional targets. Thus, a comprehensive pictureof what constitutes an effective microRNA-mRNA interaction
requires protein-level assays and the verification of functional
microRNA-target complex formation in vivo. A promisingproteomics-based validation method was reported by Plasterk. Wildtype or microRNA-mutant worms were labeled with heavy or lightisotopes, and protein fractions from these populations were mixed,
separated by 2D gel electrophoresis and analyzed by mass
spectrometry. In this differential approach, microRNA target
proteins are expected to be upregulated in the mutant. In principle,
this method should permit the direct measurement of protein levelchanges, independently of whether the corresponding mRNA is
downregulated. This method may be a particularly powerful tool forhomogeneous samples, such as cultured cells, or sorted cell types
from complex tissues. Thomas Tuschl (Rockefeller University, New
York, NY, USA) reported the co-immunoprecipitation of Argonaute
protein along with associated mRNAs from cultured human cells. In
conjunction with proteomics-based assays, the biochemical
identification of specific microRNA-target complexes should permit
the direct validation of functional endogenous microRNA-target
interactions.
microRNA-mediated inhibition of target gene
expression
microRNAs exert two general effects on target mRNAs:translational repression and a reduction in mRNA level (Carthew,
2006; Pillai et al., 2006). Both of these effects are posttranscriptional, and they involve different mechanisms. A few
animal microRNAs have sufficient complementarity to the mRNA,
which allows the mRNA to be sliced between 10 and 11 nucleotides
from the 5⬘ end of the microRNA, as is seen in traditional siRNAmediated RNA silencing (Yekta et al., 2004). However, most animal
microRNAs imprecisely match their targets and cause target mRNA
destabilization by other (non-slicer) mechanisms, such as deadenylation and decapping (Wu et al., 2006; Giraldez et al., 2006),
and/or some form of translational repression (Olsen and Ambros
1999). Most plant microRNAs have near-precise matches to theirtargets and lead to mRNA cleavage (Tang et al., 2003; JonesRhoades et al., 2006). However, translational repression can occur

DEVELOPMENT

Development 134 (9)for certain microRNA-target interactions in plants (Aukerman andSakai 2003; Chen 2004; Arteaga-Vazquez et al., 2007). Olivier
Voinnet (Institut de Biologie Moléculaire des Plantes, Strasbourg,
France) suggested that translational regulation in plants may becommon despite the prevalence of nearly perfect matches. His groupperformed mutagenesis of an Arabidopsis line bearing a green
fluorescent protein (GFP) transgene under the control of mir171.
One class of mutants isolated showed elevated levels of GFP protein
but not of GFP mRNA, as well as elevated levels of endogenous
proteins, but not of mRNAs predicted to be targeted by othermicroRNAs. The presence of this class of mutants suggests thatplant microRNAs can lead to both transcript cleavage andtranslational inhibition of target mRNAs.
Sharp discussed how the structural and base-sequence features ofan animal microRNA-target hybrid affect whether or not the
microRNA causes RNA degradation or translational inhibition.
Using microRNAs transfected into animal cells containing a set ofmutant reporter constructs, Sharp tested alternative base-pairingsand mismatches at positions 9, 10 and 11 of a microRNA that
otherwise matched at all other positions. Certain structures were
found to cause potent translational repression without affecting
mRNA level, whereas others led to mRNA reduction withouttranslational repression. These results suggest that the outcome of a
microRNA-target interaction may depend on structural features ofthe microRNA-target duplex that are recognized by microRNAassociated proteins.
Can specific microRNAs or associated 3⬘ UTRs have contextdependent effects on knockdown? Elisa Izaurralde (Max Planck
Institute, Tuebingen, Germany) used natural 3⬘ UTRs fused to aluciferase reporter to perform genome-wide RNA interference(RNAi) screens for proteins required for microRNA function, andisolated components of P-bodies, and de-adenylation and decapping enzymes (Behm-Ansmant et al., 2006). Significantly, only
certain targets were repressed by a microRNA in the absence of deadenylation or decapping enzymes, which indicates that decapping(and subsequent degradation) of de-adenylated mRNAs is theprimary mode of repression by some, but not all, microRNA-target
interactions.
Progress was reported in the search for potential mechanisms oftranslational repression by microRNAs. Ramin Shiekhattar (Centre
de Regulació Genòmica, Barcelona, Spain) reported that the
eukaryotic translation initiation factor 6 (eIF6; also known as
Itgb4bp – Mouse Genome Informatics), along with components of
the 60S ribosomal subunit, can be co-immunoprecipitated with
Argonaute 2 (Ago2; also known as Eif2c2 – Mouse Genome
Informatics) from animal cells. Moreover, RNAi knockdown ofeIF6 in animal cells, as well as in C. elegans, results in themeasurable de-repression of microRNA target expression. eIF6,
tethered to an mRNA via the Ago2-microRNA complex, could bindthe 60S ribosomal subunit, preventing the formation or stability of
80S complexes, and repressing translational initiation and/or
elongation. Marianthi Kiriakidou (from the Mourelatos group,
University of Pennsylvania, Philadelphia, PA, USA) drew attention
to amino acid similarities between a region of the Human AGO2
protein and the 7mG cap-binding domain of EIF4E. Indeed, AGO2
displayed 7mG cap-binding activity, and the mutation ofphenylalanine residues key to cap-binding activity impaired thetranslational repressive activity of AGO2 in a tethering assay.
microRNA activity can be modulated by additional protein cofactors. One such example, as discussed by Witold Filipowicz(Friedrich Meisner Institute, Basel, Switzerland), involves thehuman HuR (also known as ELAVL1 – Human Gene Nomenclature

Development 134 (9)Database) protein, which is exported from the nucleus of liver cells
in response to stress. This Elav-family RNA-binding protein binds
to an element in the 3⬘ UTR of CAT-1 (also known as SLC7A1 –Human Gene Nomenclature Database) mRNA and de-represses
CAT-1 protein production by interfering with the repressive activityof miR-122 (MIRN122A) (Bhattacharyya et al., 2006). miR-122
activity leads to the sequestration of CAT-1 mRNA in P bodies,
whereas, upon HuR binding, CAT-1 mRNA exits the P bodies and istranslated, perhaps via the displacement of the microRNA from themRNA. Similarly, Antonio Giraldez (Yale School of Medicine, New
Haven, CT, USA) reported how the Nanos 3⬘ UTR mediates therepression of a mir-430 family microRNA sensor in zebrafish
somatic cells, but not in the germline. The effect is due to a propertyof the 3⬘ UTR outside of the miR-430 sites and is consistent with thepresence of a germ-cell-specific factor that blocks miR-430 activity
in the context of some 3⬘ UTRs, but not others (Mishima et al.,2006).
One area that is beginning to garner more attention is the intrinsic
secondary structure of the mRNA in the vicinity of the microRNAtarget site, and how this could affect the accessibility of the microRNAto its complementary sequences (Robins and Padgett, 2005; Zhao etal., 2005). Bartel commented that two features of functional sites –A/U-rich sequences surrounding the site and preferential placementof the more-effective sites at the ends of the 3⬘ UTR – could influence
the accessibility of the microRNA-binding site in the 3⬘ UTR. Renée
Schroeder (Max Perutz Laboratories, Vienna, Austria) reported resultsfrom RNA-induced silencing complex (RISC)-binding and targetcleavage experiments performed in collaboration with Javier Martinez(IMBA, Vienna, Austria). By careful quantitation of the velocity of
the RISC-cleavage reaction, and the biochemical verification ofpredicted target secondary structures, a strong effect of target
accessibility on RISC binding was convincingly demonstrated. It will
be exciting to see if these biochemical analyses can be adapted to thenon-cleaving activity of microRNA-guided RISC, and whether the
influence of a target secondary structure can also be observed fornatural 3⬘ UTR substrates.
Gene regulation by piRNAs and endogenous
siRNAsRecent advances have illuminated the surprisingly diverse modes ofsmall RNA-guided gene regulation by Argonaute-based
mechanisms (Fig. 1). The Piwi class of Argonaute proteins isessential for spermatogenesis in mice and for gametogenesis in flies(Cox et al., 1998; Kim, 2006); in the past year or so, a new class of
small RNAs – piRNAs – have been found to be associated with Piwi
family proteins (Saito et al., 2006; Vagin et al., 2006; Grivna et al.,2006; Aravin et al., 2006; Girard et al., 2006; Lau et al., 2006).
Although piRNA function in gametogenesis is still unclear, Gregory
Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,
USA) and Mikiko Siomi (University of Tokushima, Tokushima,
Japan) reported progress in understanding the biogenesis andactivity of Drosophila repeat-associated siRNA (rasiRNAs), which
are similar to mammalian piRNAs in many respects.
Hannon presented results from studies on the biogenesis andfunction of a cluster of rasiRNAs from the flamenco locus, atransposon-rich heterochromatic locus on the fly X chromosome thatserves as a master control locus to silence gypsy, ZAM and othertransposons scattered around the genome. The flamenco locus
spawns rasiRNAs that presumably interact with, and silence,homologous transposon sequences throughout the genome. Featuresof these rasiRNAs reveal aspects of their biogenesis via a ‘pingpong’ mechanism that occurs without the involvement of Dicer

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1638 MEETING REVIEW

activity and results in the amplification of rasiRNAs. These results
suggest that, one way to generate rasiRNAs is by Slicer-mediatedcleavage of target transcripts.
Siomi presented similar findings for the Suppressor of stellate
locus, which generates rasiRNAs that act in an Auberginedependent manner to silence Stellate (also known as SteXh –FlyBase) mRNA/protein in fly testes. Sarah Elgin (Washington
University, St Louis, MO, USA) showed that Drosophila Piwi and
Aubergine are required for repeat-induced gene silencing. In acollaborative study with Haifan Lin (Yale University, New Haven,
CN, USA), Piwi was found to interact with Heterochromatin protein
1 (HP1) in yeast two-hybrid assays, which implicates Piwi, andperhaps piRNAs, in heterochromatin formation.
Histone modification is crucial to the regulation andstabilization of gene expression, and small RNAs play a central
role in this process. Shiv Grewal [National Institutes of Health(NIH), Bethesda, MD, USA] provided evidence for the existenceof a self-reinforcing loop mechanism, in which heterochromatincomponents stably tether RNAi machinery across chromosomal
domains that contain certain repeats. These heterochromatinbound RNAi factors may selectively produce repeat siRNAs inorder to fend off future invasion by similar sequences (Grewal andJia, 2007). Grewal introduced a multi-enzyme silencing effector
complex in Schizosaccharomyces pombe (termed SHREC) thatconnects the RNAi pathway to chromatin remodeling. SHREC
binds the heterochromatin platform established by the RNAi
machinery and effects transcriptional gene silencing via theactivities of the histone deacetylase Clr3 and a SNF2 family
ATPase Mit1 (Sugiyama et al., 2007). Martin Gorovsky(University of Rochester, Rochester, NY, USA) illustrated theimportance of a class of small RNAs (scanRNAs) for proper
conjugative reproduction in the protist T. thermophila. scanRNAs
direct histone modifications in the newly replicated ‘somatic’nucleus.
Plants also use similar mechanisms to maintain heterochromatin,
as discussed by David Baulcombe (Sainsbury Laboratory, Norwich,UK) and Marjori Matzke (Gregor Mendel Institute, Vienna, Austria)(Fig. 1). Matzke reported the identification of loci that are
methylated and silenced by endogenous siRNAs in an RNA
polymerase (Pol) IVb-dependent manner. The Pol IVb-silenced lociinclude repeats, long terminal repeats (LTRs) of retrotransposons,and DNA transposons. Many of the Pol IVb-silenced loci also
contain short internal tandem repeats, which are thought to help
sustain RNA-dependent RNA polymerase (RdRP) activity ingenerating double-stranded RNA (dsRNA), from which, siRNAs are
generated through Dicer activity.
Steven Jacobsen (University of California, Los Angeles, LA,
USA) presented genome-wide profiles of DNA methylation (Zhang
et al., 2006) and histone H3 lysine 27 (H3K27) trimethylation (me3)in Arabidopsis. H3K27me3 marks in animals span large regions ofdevelopmentally important loci, such as the Hox clusters and certain
gene promoters, and are regulated by three Polycomb repressor
complexes; Polycomb repressive complex 1 (PRC1), PRC2, andPho-repressive complex (PhoRC). Plants, however, only possess the
PRC2 complex, and this might explain why H3K27me3 tends to be
restricted to small regions (within 1 kb) in plants. H3K27me3 marks
are found in the main body (not at the promoter or 3⬘ end) of genes
on euchromatic chromosomal arms. H3K27me3 genes tend to be
expressed in a tissue-specific manner and probably havedevelopmental roles. Curiously, the H3K27me3 marks do notcorrelate with the presence of small RNAs, nor with DNA
methylation.

MEETING REVIEW 1639

Worms also carry out ongoing silencing of endogenous genes by
RNAi (Lee et al., 2006). Endogenous worm siRNAs are probably
generated by mechanisms similar to those that produce secondarysiRNAs, which induce long-lasting, heritable silencing (Sijen et al.,2007; Pak and Fire, 2007). Andrew Fire’s (Stanford University, Palo
Alto, CA, USA) results indicate that the majority of secondarysiRNAs are produced by de novo synthesis of short RNAs by RdRp.
Craig Mello (University of Massachusetts, Worchester, MA, USA)reported that endogenous siRNAs are also produced de novo by
RdRp and that they depend on the activity of Dicer related helicase(DRH-3). Mello suggested that much of the worm genome is subject
to ongoing, epigenetic and heritable gene silencing, and, therefore,
natural selection could maintain favorable epigenetically determinedvariations in gene expression.
Evolution of small regulatory RNA function
Several plant and animal microRNAs are highly conserved within
their respective kingdoms (Pasquinelli et al., 2000; Axtell andBartel, 2005), indicating that microRNA-based gene regulation
emerged early during the evolution of both plants and animals,
perhaps in conjunction with the acquisition of complex multicellularforms. The phylogenetic distribution of conserved microRNAs in
animals (Sempere et al., 2006; Prochnik et al., 2007) indicates thatat least 27 of the conserved microRNA families arose in the ancestorof all bilaterians (the ‘urbilaterian’), whereas perhaps just threeconserved families evolved earlier, in an ancestor to bilaterians andcniderians. The rapid expansion of microRNA number and type inan urbilaterian ancestor suggests that these post-transcriptional
regulators played a role in the adaptive diversification of pre-existingtranscriptional and cell-cell signaling pathways required for theevolution of complex and diverse organs and body plans.
Many microRNAs and their target interactions appear to be
rapidly evolving, suggesting an ongoing potential for microRNAs
to drive animal and plant diversity. James Carrington (Oregon State
University, Corvallis, OR, USA) revealed how his laboratory
recently identified non-conserved microRNA loci in Arabidopsis,
supporting the view that new plant miRNA loci may evolve from theinverted duplication of founder loci, producing a hairpin RNA.
Dicer-like 4 (DCL4)-processing of the new hairpin leads to thegeneration of siRNAs, and microRNAs would subsequently evolve
via DCL1 activity as the hairpin RNA accumulates mismatches.
Carrington noted that, in plants, the recently evolved microRNAs
seem to be far less likely to target transcription factors thanconserved microRNAs (Fahlgren et al., 2007); indeed, some do nothave any predicted targets, suggesting that they could be on an
evolutionary path to elimination. Carthew found that Drosophila
species-specific microRNAs are expressed at relatively low levels
and, hence, some of these microRNAs could be evolutionarily
neutral. Rajewsky pointed out that some of the non-conserved
targets of conserved microRNAs could be similarly neutral. These
exciting ideas suggest that organisms contain a vast reservoir ofpotentially important microRNA-target regulatory interactions
poised for natural selection.
Phil Zamore (University of Massachusetts, Worchester, MA,
USA) and Narry Kim (Seoul National University, Seoul, Korea)
described aspects of microRNA biogenesis that profoundly affectmicroRNA expression and activity, and hence are crucial factors inmicroRNA function and evolution. Zamore reported structuralfeatures of microRNA precursors in flies that govern the sorting ofa microRNA to the correct Argonaute effector complex. Kim notedthat most mammalian microRNAs are encoded in the sense strand
of introns, often within protein-coding genes, and reported on

DEVELOPMENT

Development 134 (9)studies of the mechanistic and regulatory relationship between premRNA splicing of the host gene and DROSHA (also known as
RNASEN – Human Gene Nomenclature Database)-mediatedprocessing of the intronic microRNA.
Are endogenous siRNAs broadly conserved in animals? The
comparative genomics of RNAi pathways and proteins between
plants and animals indicate that many endogenous siRNAs areproducts of RdRPs; interestingly, among animals, only nematodes
are reported to contain such genes (Cerutti and Casas-Mollano,2006). The known features of mammalian piRNAs and Drosophila
rasiRNAs from the flamenco locus do not support the possible
existence of a precursor dsRNA or an RdRP-based mechanism.
Even the deep-sequencing of small RNAs from mouse ES cells(reported by Sharp) did not reveal endogenous siRNAs. However,Baulcombe issued a cautionary tale of the unicellular algae
Chlamydomonas reinhardtii, which lacks an RdRP gene in its
genome, but expresses a class of endogenous siRNAs that bears thehallmarks of endogenous siRNA clusters that are seen in plants andworms.
Conclusion
The field of small regulatory RNAs is enjoying rapid progress. We
continue to be surprised by the diversity of small RNAs and theirregulatory mechanisms, and it is likely that the complexity of these
phenomena will continue to blossom in the coming months andyears. Some of the immediate challenges are to elucidate thedevelopmental roles of piRNAs, and the mechanisms of biogenesisof piRNAs, rasiRNAs and endogenous siRNAs. Precisely how dosmall RNAs mediate the epigenetic regulation of gene expression?
By what mechanisms do microRNAs influence the translational
efficiency and stability of target mRNAs? What factors, specific or
non-specific, modulate microRNA target recognition andrepression? Can microRNAs activate the expression of a target? One
challenge for the future will be to understand the evolutionary
mechanisms that have fixed the entire 21 nucleotide sequence of
certain microRNAs for hundreds of millions of years (Pasquinelli etal., 2000; Sempere et al., 2006; Prochnik et al., 2007). The answers
to these and other questions may emerge when we return to the
Keystone Symposium next year.
We thank D. Bartel and S. Grewal for organizing the meeting program; all the
speakers for their excellent presentations and for permitting us to discuss theirunpublished results; and J. Carrington, N. Lau and J. Brennecke for commentson the manuscript. We apologize to those whose work could not be discussed
owing to space constraints. V.A. is supported by grants from NIH. X.C. is
supported by grants from NIH and NSF.
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