Developmental Cell 10, 419–424, April, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.devcel.2006.03.005

Demystifying Small RNA Pathways

Amy E. Pasquinelli*
Department of Biology
University of California, San Diego
La Jolla, California 92093

The Keystone Symposium entitled ‘‘RNAi and Related
Pathways,’’ organized by Craig Mello (University of
Massachusetts), Phillip Zamore (University of Massachusetts) and James Carrington (Oregon State University), was held in Vancouver, British Columbia. The
meeting participants reviewed recent reports andpresented new advances in our understanding of thewidespread role of small noncoding RNAs in gene
regulation.

The first hints of unconventional homology-mediated silencing phenomena surfaced during the early and mid
1990s in plants, fungi, and nematodes (Guo and Kemphues, 1995; Napoli et al., 1990; Romano and Macino,
1992; Smith et al., 1990; van der Krol et al., 1990). However, it was not until the end of the decade that themechanism and broad utility of RNA interference(RNAi)-related gene regulation started to be fully realized. The microRNA (miRNA) field also quietly existed
as a single worm gene throughout most of the 1990’s(Lee et al., 1993). By the turn of the century, though,RNAi and miRNAs had caught the attention of many researchers intent on exploring the broad biological andtherapeutic potential of gene regulation mediated by
w22 nucleotide noncoding RNAs. As keynote speaker
David Baulcombe suggested, the romance period during the early years of the twenty-first century has now
developed into a marriage state for investigators ofRNAi and related pathways with the mysteries giving
way to tangible complexities. Overlap and redundancy
in regulatory mechanisms and small RNA guides, theunknown abundance and types of small RNAs, and expanded RNAi/miRNA factor gene families were repeating themes at the 2006 RNAi and Related Pathways Keystone Meeting. The intertwined nature of different typesof small RNA pathways and other forms of gene regulation gave way to diverse topics in most plenary sessions. In this meeting review, I highlight some of theoutstanding talks in a thematic rather than chronologicalorder (Figure 1).
RNAi Mechanism
The long sought mechanism of RNAi has matured to theatomic resolution. This process uses small interfering
RNAs (siRNAs) to guide RNA-induced silencing complexes (RISC) for cleavage of complementary target
messenger RNAs (mRNAs). The endonucleolytic cleavage, or ‘‘slicing,’’ of the target mRNA usually severs
the phosphodiester bond that links the tenth and eleventh nucleotides base-paired to the siRNA. RISC is*Correspondence: apasquin@ucsd.edu

Meeting Review

a multiprotein complex that includes Argonaute proteinsat its core. Identification of the RISC factor responsible
for slicing target mRNAs was greatly aided by structural
studies of Argonaute homologs. The PIWI domain common to all Argonaute proteins contains an RNaseH fold
(Parker et al., 2004; Song et al., 2004), and predicted catalytic residues within this motif are required for RISC
slicer activity (Liu et al., 2004; Meister et al., 2004). David
Barford (Institute of Cancer Research, London) and Dinshaw Patel (Memorial Sloan Kettering Cancer Center)each summarized their insights into the function of the
PIWI domain based on impressive independent crystal
structures of the Archaeoglobus fulgidus Piwi protein
bound to an siRNA-like duplex (Ma et al., 2005; Parker
et al., 2005). The structures show that the PIWI domain
places the 50 half of the guide for solvent exposure, likely
to accommodate target recognition, and positions thescissile phosphate bond between target strand nucleotides 10 and 11 into the nuclease active site. Additionally, unpairing of the first nucleotide of the guide strand
and anchoring by PIWI domain residues are importantfor efficient cleavage activity. These models refine ourunderstanding of the molecular mechanism of RISC
activity and may contribute to the design of optimized
siRNAs and small molecules that could selectively modulate Argonaute function.
The slicer function of some Argonaute proteins canplay dual roles in the RNAi pathway. In addition to cleaving the target strand of an mRNA-siRNA duplex, the discarded passenger strand siRNA can also be sliced inhalf by Argonaute (Matranga et al., 2005; Miyoshi et al.,2005; Rand et al., 2005). Mikiko Siomi (University ofTokushima, Japan) and Phillip Zamore (University of
Massachusetts) each showed that Drosophila Ago2 cancleave the passenger strand of an siRNA duplex and that
this event facilitates RISC maturation. It remains to be
explained how cleavage of the passenger strand favors
its displacement from Ago2.
In C. elegans and plants, the potency of RNAi is
boosted by the amplification of siRNA guides and thesystemic spread of the silencing signal. Craig Mello (University of Massachusetts) presented evidence that the
Argonaute member RDE-1 (RNAi defective) is required
to initiate silencing induced by double-stranded RNA(dsRNA), but other family members perform the downstream job of using amplified secondary siRNAs tomaintain the silenced state. Worms have over 25 Argonaute genes, and the heroic creation of strains combining deletion mutations in six of them was necessary toassign some of these genes roles in the RNAi pathway.
Interestingly, overexpression of single Argonaute family
members rescues the RNAi-defective phenotype of the
sextuple mutant, even if the gene lacks catalytic residues important for slicer activity. The issue of whether
worms produce typical RISC cleavage products is yet
to be resolved. Craig Hunter (Harvard University) updated meeting attendees on the genes that emerged
from the clever screen preformed by his lab to identify
mutants specifically defective in systemic RNAi in C. elegans. The SID-1 (systemic RNAi defective) multipass

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Figure 1. Expression and Function of Small
RNAs
Precursors of small RNA guides include
miRNA primary transcripts, longer doublestranded RNAs for generating siRNAs, and,in the case of some rasiRNAs, the substratetranscripts have not been characterized. Often multiple processing events and, at leastin plants, modifications help generate thesmall w22 nt guide RNAs that assemble
with protein partners. Some plant miRNAs set the phase for the production of ta-siRNAs. Typically, miRNAs and siRNAs inhibit mRNA and viralgene expression by promoting RNA degradation or translational repression. The rasiRNAs primarily direct silencing of transposons and repeat
elements by guiding chromatin remodeling and modifications of histones and DNA at homologous loci.

transmembrane protein appears to mediate both importand export of the systemic signal. Expression of thisprotein in Drosophila S2 cells enables uptake ofdsRNAs, preferably 50 base pairs or longer (Feinberg
and Hunter, 2003). This observation may be relevant
for defining the nature of the endogenous RNAi signalthat spreads in C. elegans.

Murine and primate polyomaviruses express late-phase
miRNAs that inhibit early viral transcript accumulation to
reduce antigen production and optimize infectivity inanimals. Remarkably, the murine polyomavirus encodes
miRNA genes that are conserved in function but not insequence or genomic positions with the primate-likepolyomaviruses.

Silencing and Defense
The complex interplay between RNAi factors and the viral proteins that evolve to thwart them was a prevalent
theme at the meeting. Multiple silencing pathways
have been well established in plants as defense mechanisms against viruses. In his keynote address, David
Baulcombe (The Sainsbury Laboratory) reported a newmechanism viruses can use to evade silencing. Someluteoviruses encode a silencing suppressor that has
E3 ubiquitin ligase activity, which may tag Argonaute
proteins for destruction by the proteosome, crippling
the plant’s RNAi defense mechanism. Olivier Voinnet (Institut de Biologie Moleculaire des Plantes) explained
a plant defense mechanism whereby DCL2 (Dicer like)can substitute for DCL4 when it is inhibited by the Turnip
Crinkle Virus p38 gene product. Additional evidence
that variable and often combined Dicer activities are involved in mounting plant silencing defenses supports
his proposal that diversification of Dicer proteins inplants may be driven by adaptation to viral suppressors.
Herve Vaucheret’s (Insititut Jean-Pierre Bourgin) analysis of combinations of Dicer mutations revealed that
there are redundancies and, surprisingly, antagonistic
effects on small RNA pathways in this expanded genefamily in plants. Thus, the dependence on distinct small
RNA pathways for gene regulation and viral defense has
resulted in expanded numbers and complex interplayfor plant silencing factors.
Animal viruses also can foil or utilize RNAi pathways
for their own selfish needs. Shou-Wei Ding (Universityof California, Riverside) demonstrated that the B2 protein of the Flock House Virus (FHV) blocks the RNAi defense systems in Drosophila S2 cells and in C. elegans(Lu et al., 2005). Additionally, adult flies with mutationsin the RNAi factors DCR-2 or R2D2 exhibit enhanced disease susceptibility and shortened lifespans following
viral infection, supporting Ding’s conclusion that RNAi
significantly contributes to innate immunity strategies
in invertebrates. Animal viruses can also harness thecellular RNAi machinery to optimize infectivity. Chris
Sullivan (University of California, San Francisco) fromDon Ganem’s lab described the contribution of polyomavirus miRNAs to infection (Sullivan et al., 2005).

RNAi for Therapy
Taking a cue from endogenous viral defense systems,
several groups are developing RNAi strategies to combat viral infections and treat human diseases. Judy Lieberman (Harvard Medical School) presented her lab’s
recent demonstration that siRNAs targeted against lethal Herpes Simplex 2 Viral genes offer potent protection
from infection in mice (Palliser et al., 2006). Respiratory
viruses are a major target for RNAi therapies being developed by Antonin de Fougerolles and his group at
Anylam Pharaceuticals, Inc. Hope for restoring vision
loss due to ocular neovascularization was offered by
Pamela Pavco and her group at Sirna Therapeutics,
Inc. Injection of siRNAs that target vascular endothelial
growth factor receptor 1 (vegfr1) caused downregulation of vegfr1 mRNA levels and significant reduction in
neovascularization in a mouse model system (Shenet al., 2006). Rene Bernards (Netherlands Cancer Institute) presented the large-scale RNAi screens his groupis using to study cancer-related genetic pathways. A
primary goal is to use their short hairpin RNA (shRNA)
vector libraries to identify genes whose loss of function
makes cancer cells selectively drug sensitive.
Biological Function of miRNA Pathways
In his keynote address, Victor Ambros highlighted thecomplexities involved in defining biological roles for
specific miRNAs. Many miRNAs belong to families thatcan share functions in convergent, divergent, or linear
regulatory pathways. Three different C. elegans miRNAs
share sequence homology with let-7 miRNA but exhibit
different temporal expression patterns from let-7 andshow functional redundancy among themselves but
not with let-7 in target specification (Abbott et al.,2005). A new role for the founding miRNA gene, lin-4,
was presented by Frank Slack (Yale University). He
showed that lin-4 miRNA regulates the maturation ofa worm neuron and raised the therapeutic possibility
that modulating miRNA activity may be a way to reset
neuronal identity.
The surprising dearth of phenotypes that often result
from loss of a single miRNA can be at least partiallyexplained by the multilayered and compensatory

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mechanisms that regulate development. Richard Carthew (Northwestern University) described the complex
interplay among transcriptional, posttranscriptional,
and posttranslational systems that respond to epidermal growth factor receptor signaling to promote photoreceptor differentiation in the Drosophila eye (Li andCarthew, 2005). Mir-7 functions in this pathway, butloss of the miRNA has little negative effect on eye development because the other layers of regulation compensate. The utilization of redundant gene regulatory mechanisms to assure proper developmental programs was
also a theme of Eric Lai’s (Sloan Kettering Institute) presentation. Ectopic expression of the Drosophila miRNAiab-4 induces homeotic transformation of halteres into
wings, due to direct downregulation of Ultrabithorax.
However, as this miRNA gene appears to be dispensable for viability, back-up mechanisms likely exist to
prevent substantial misexpression of iab-4 targets (Ronshaugen et al., 2005).
William Theurkauf (University of Massachusetts) demonstrated the important lesson that phenotypes resulting from defective RNAi factors do not always directly
relate to small RNA pathways. Axis specification in Drosophila oocytes is regulated by asymmetric RNA localization. This process is disrupted by mutations in thearmitage, spindle-E and aubergine genes, which encode
helicases and an Argonaute protein important for efficient RNAi (Cook et al., 2004). Surprisingly, second mutations in the ATR and Chk2 kinase DNA damage response genes rescue the asymmetry but not the RNAi
defects in all three mutations. Thus, the critical functionof the RNAi pathway during oogenesis is to suppressa DNA damage response, which in turn disrupts axis
specification.
Cell lineage specification and axis formation in Zebrafish also appears possible without miRNA function(Giraldez et al., 2005). Instead, miRNAs are required toundergo normal morphogenesis. The first detectable
miRNA, mir-430, in developing zebrafish was proposedby Antonio Giraldez from Alexander Schier’s lab (Harvard University) to play a role in eliminating maternal
mRNAs at the mid-blastula transition. Many of the genes
upregulated in the absence of mir-430 are maternally inherited and contain predicted mir-430 target sites. Giraldez presented evidence that miRNAs may accelerate
the decay of target mRNAs by promoting their deadenylation. Ronald Plasterk (Hubrecht Laboratory, The Netherlands) reviewed some of his group’s impressive in situ
analyses of miRNA expression patterns in zebrafish embryos (Wienholds et al., 2005) and discussed their progress in defining functions for specific miRNAs. Abouthalf of the known zebrafish miRNAs are expressed inbrain tissues. Although morpholino knockdown of single
miRNAs often produced no detectable phenotypes, thisstrategy has implicated some miRNAs in specific biological functions, such as touch perception. These talks
vividly demonstrated that zebrafish has emerged asa powerful system to understand the function of vertebrate miRNAs.
Biogenesis of miRNAs
In animals, Drosha and its partner DGCR8, also called
Pasha, recognize and process miRNA hairpin precursors from longer primary transcripts. V. Narry Kim (Seoul

National University) defined RNA features important forthis maturation step. Optimal processing requires
a lower stem region followed by a single-stranded region at the base of the precursor stem, which includes
three helical turns and often is interrupted by central unpaired bases; a terminal loop is apparently inessential.
These parameters may aid in the identification of additional Drosha substrates and in the design of siRNA producing vectors.
Modification of plant miRNAs appears critical for their
existence. Xuemei Chen (University of California, Riverside) showed that Arabidopsis HEN1 encodes a methyltransferase that modifies the 20 hydroxyl of the miRNAterminal 30 nucleotide (Yang et al., 2006; Yu et al.,2005). This methyl group may protect miRNAs froma 30 end uridylation activity (Li et al., 2005). Potential
HEN1 homologs exist in animals, but methylation of animal miRNAs has not been reported.
Mechanism of miRNA Function
A common mechanism for how miRNAs function was
not resolved at this meeting and, in fact, may not exist.
As Victor Ambros pointed out in his keynote address,
multiple pathways may be available for miRNAs to effectively inhibit target gene expression. Independent studies by Witold Filipowicz (Friedrich Miescher Institute for
Biomedical Research) and David Humphreys from
Thomas Preiss’s group (Victor Chang Cardiac ResearchGroup) led to the conclusion that miRNA regulation ofsome targets is through inhibition of translation initiation(Humphreys et al., 2005; Pillai et al., 2005). The mechanism by which miRNA complexes interfere with translation initiation is yet to be elucidated. Filipowicz alsopresented a specific example of an endogenous miRNAtarget that appears to be regulated at the translational
level. Expression of mammalian cat-1 (cationic aminoacid transporter) is subject to multiple levels of regulation (Hatzoglou et al., 2004), which now appears to include a miRNA pathway. Filipowicz reported that miR122 inhibits CAT-1 protein expression, but not mRNAstability, in liver cells. Interestingly, miR-122-mediatedrepression of CAT-1 appears to respond to cellularstress.
The eventual fate of many mRNAs targeted for regulation by the miRNA pathway appears to be degradation.
Microarray experiments presented by several speakers
confirmed that expression of miRNAs is related todownregulation of many mRNAs with predicted target
sites. Additionally, regulation of genetically defined targets of the founding miRNA genes in C. elegans involves
mRNA destabilization (Bagga et al., 2005). To uncovermechanisms by which miRNA targeting can lead tomRNA degradation, Alan Zahler and Guoping Gu (University of California, Santa Cruz) partially purified alin-4 miRNA complex from C. elegans. When presented
with a substrate resembling its natural partially complementary target, the fraction containing the lin-4 miRNP
exhibited cleavage activity but the RNA products were
not characteristic of slicer action. Zahler suggested
that a yet to be identified nuclease may be tethered tothe target by this miRNP.
miRNAs can guide the generation of other small RNA
species. James Carrington (Oregon State University) described an unexpected function for several Arabidopsis

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miRNAs. These miRNAs guide the cleavage of target
transcripts to set the phase for trans-acting siRNAs(ta-siRNAs) (Allen et al., 2005). Following cleavage, theta-siRNA substrates are converted to dsRNAs and processed by Dicer to yield 21 nucleotide ta-siRNAs thatthen target other genes for negative regulation.
miRNA Targets
Significant percentages of animal genes seem to be direct targets of miRNA regulation. David Bartel (MIT) reported that about one-third of human genes are underselective pressure to maintain pairing with miRNA
seed sequences—generally nucleotides 2–7 from the
50 end of the miRNA (Lewis et al., 2005). Comparisonof mouse tissue-specific expression patterns of miRNAs
and predicted targets led to the conclusions that conserved targets tend to be preferentially downregulated
in miRNA-enriched tissues and that genes highly coexpressed with specific miRNAs are depleted of cognate
target sites (Farh et al., 2005). Bartel proposed that theextensive depletion of these sites, which are only 7 nt
in length, indicates that such sites are often sufficientfor miRNA-directed repression and implies widespreadnonconserved targeting. Similarly, Nikolaus Rajewsky(New York University) also reported that a high fractionof the human genome is targeted by miRNAs and that
there is an inverse correlation between human tissuespecific miRNAs and target mRNAs predicted by his algorithm PicTar (Krek et al., 2005; Sood et al., 2006). He
also presented a computational tool that directly correlates 30 UTR motifs with changes in mRNA levels assayed by genome wide microarray experiments. To validate predictions made by an updated version of the
PicTar algorithm, Rajewsky turned to C. elegans as
a robust experimental system, and presented a platform to test endogenous 30 UTR-mediated regulation.
At least 10% of worm genes have conserved miRNAtarget sites. Some of the newly predicted targets of
the let-7 miRNA exhibited reporter gene expression
patterns that are consistent with direct regulation by
this miRNA (Lall et al., 2006).
In contrast to the apparent broad specificity of animal
miRNAs, plant miRNAs are relatively selective of their direct targets. Detlef Weigel (Max Planck Institute) reminded meeting participants that plant targets do include genes with up to five mismatches. Parametersthat restrict miRNA-directed cleavage of plant targets
include greater than 70% free energy of perfect match,no mismatch at the cleavage site and only one within
the seed pairing region, and no more than two consecutive mismatches with the 30 end of the miRNA (Schwab
et al., 2005). The Weigel lab is employing these rules tocreate artificial miRNAs for targeting specific membersof related gene families to study the global effects on
gene regulatory networks. He concluded that plant
miRNAs have specific effects on a small number of direct targets to orchestrate coordinated responses thatare highly connected.
New Small RNAs
Discovery of new small RNAs has skyrocketed in partdue to the introduction of ‘‘deep sequencing’’ technologies this past year. In addition to probing the small RNA
depths of Arabidopsis, David Baulcombe (The Sains-bury Laboratory) also explored Chlamydomonas. His
group found evidence of possible miRNAs in this green
algae species—perhaps the first demonstration of thissmall RNA class in a unicellular organism. In C. elegans,over 370,000 sequencing reads generated by David Bartel’s group (MIT) have revealed new miRNAs and a potentially novel class of small RNAs.
Novel small RNA pathways regulate transposon silencing in Drosophila. Phillip Zamore (University of Massachusetts) reported that retrotransposon silencing in
Drosophila relies on sets of factors and small RNAs
that distinguish the mechanism from that utilized by
the siRNA and miRNA pathways. Using tiling microarrays, the Zamore group demonstrated that the Y-linked
Suppressor of Stellate (Su(Ste)) locus, produces small
RNAs of 25–27 nucleotides to silence the Stellate geneon the male X chromosome. Curiously, these repeatassociated RNAs (rasiRNAs) are only generated fromone strand in an unphased pattern, which prompts theyet to be answered question of how they are generated.
Transcriptional Silencing
The silencing role of small RNAs is not limited to posttranscriptional regulation of other RNAs. Compelling evidence that small RNAs and the RNAi machinery control
DNA and chromatin activities was presented in the final
session of the Keystone meeting. Sarah Elgin (Washington University) defined features that initiate heterochromatin formation in Drosophila melanogaster. A cleverscreen for position effect transgene silencing helped uncover the 1360 transposon element on chromosome
four that directs heterochromatin formation across distances up to 10 kb away. Both proximity to a copy oftransposon 1360 and the RNAi machinery were demonstrated as necessary for efficient heterochromatin formation at a silenced reporter. siRNAs corresponding
to the 1360 transposon may provide a local signal forheterochomatin formation.
A role for the RNAi machinery in heterochromatin formation extends to unicellular organisms. Studies in thefission yeast S. pombe have revealed that RNAi factors
and small RNAs are linked to the recruitment of heterochromatin proteins and histone modifying enzymes at
specific genomic loci. Shiv Grewal (National Institutesof Health) showed that RNAi mechanisms and DNA
binding proteins are important for establishing heterochromatin at the mating locus (Yamada et al., 2005).
He raised the question of how the cell generates transcripts for siRNA production if the homologous locus
is thick in heterochromatin. There appears to exist a specialized mechanism that promotes transcription of heterochromatic repeats, which is essential for generationof siRNAs. The siRNAs may provide the inheritable element needed to specify the epigenetic state.
Concluding Remarks
Despite the initial slow momentum in recognizing the existence of small RNAs and their diverse functions, thisnew field has amassed a faithful and rapidly expanding
following of researchers intent on demystifying RNAi
and its related pathways. This goal is challenged by
the overlapping, redundant, and compensatory featuresof the biological pathways regulated by small RNAs.
Deep sequencing and tiling array technologies indicate

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that small RNAs are rich in numbers and types, fulfilling
the promise that genomes hold secrets far beyond what
we can currently project. Computational methods have
proven invaluable for predicting small RNAs and theirregulatory targets. Sophisticated bioinformatic approaches, founded on experimental evidence, in combination with rigorous genetic studies will be requisite for
guiding our future attempts to assign specific small
RNAs and their protein cohorts into complex gene regulatory networks.
Acknowledgments
I thank T. Nilsen and K. Massirer for critical reading of the manuscript
and meeting participants for permitting inclusion of their work. I
apologize to presenters who were not referenced due to space limitations. My lab is supported by the National Institutes of Health(GM071654-01) and the Searle Scholars, Peter Gruber, and V Foundations.
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