Ameres and Fukunaga Silence 2010, 1:8
http://www.silencejournal.com/content/1/1/8

REVIEW

Open Access

Riding in silence: a little snowboarding, a lot ofsmall RNAs
Stefan L Ameres*, Ryuya Fukunaga*
Abstract
The recent symposium, RNA silencing: Mechanism, Biology and Applications, organized by Phillip D. Zamore(University of Massachusetts Medical School) and Beverly Davidson (University of Iowa), and held in Keystone,
Colorado, brought together scientists working on diverse aspects of RNA silencing, a field that comprises amultitude of gene regulatory pathways guided by microRNAs, small interfering RNAs and PIWI-interacting RNAs.
Review
From 14 to 19 January 2010, small RNAs once again
attracted the attention of more than 500 attendees tosymposium on RNA silencing: Mechanism, Biology andApplications, held in Keystone, Colorado. In the midstof the breathtaking panorama of the Rocky Mountainsummits, exciting and rapidly evolving science mixed
with superb riding and skiing created a stimulating
atmosphere for discussions on the mechanisms, biology
and applications of RNA silencing.
Biogenesis of small RNAs

A much-discussed topic at the meeting was the mechanisms and regulation of micro (mi)RNA biogenesis. In
her keynote address, Narry Kim (Seoul National University) elaborated on the effect of pre-miRNA uridylation.
She reported that the terminal uridylyl transferase,
TUT4, acts on the 3’ termini of pre-miRNAs to suppress
pre-miRNA processing [1,2]. Although this process
requires the scaffold protein Lin-28 to recruit the
TUTase to specific pre-miRNAs, uridylation is not limited to these pre-miRNAs. Whereas oligo-uridylylation
inhibits pre-miRNA dicing, mono-uridylylation canenhance pre-miRNA processing by human and fly Dicer
proteins in vitro, suggesting that post-transcriptional
pre-miRNA modification can have a versatile effect onprocessing. Surprisingly, the addition of one or a few
additional nucleotides to the 3’ end of a pre-miRNA did
not alter the 3’ end of the miRNA excised from the 5’* Correspondence: stefan.ameres@umassmed.edu; ryuya.
fukunaga@umassmed.edu
Department of Biochemistry and Molecular Pharmacology, Massachusetts
Medical School, 364 Plantation Street, Worcester, MA 01605, USAside of the stem. To explain these data, Kim proposedan alternative model to the PAZ-anchored molecularruler model that emerged from the structure of Giardia
Dicer [3]. She suggested that Dicer proteins in higher
eukaryotes measure mainly from the 5’ end of a premiRNA, rather than from its 3’ end.
Jennifer Doudna (University of California, Berkeley)described the first structural insights into human premiRNA processing and loading by electron microscopy[4]. She presented biochemical cross-linking studies
employing a recombinant human Dicer-TRBP (trans activation response RNA binding protein) complex. These
data suggest that the human proteins, ‘sense’ the thermodynamic asymmetry of the ends of a small RNA duplex, as
was originally proposed for their fly counterparts, Drosophila Dicer-2 and R2D2 [5]. For both the human and fly
proteins, the double-stranded (ds)RNA binding partner ofDicer binds to the more stable end of a small interfering(si)RNA duplex, directing the preferential loading into
Argonaute of the siRNA strand, whose 5’ end is less stably
paired. PACT (protein activator), the other dsRNA-binding partner of human Dicer, showed similar behavior inthese studies. Despite this similarity, the two dsRNA-binding partners of Dicer had opposite effects on pre-miRNAprocessing: TRBP activated dicing thus increasing turnover, whereas PACT inhibited dicing. Thus dsRNA-binding dicer partner proteins may tune Dicer activity.
Dinshaw Patel (Memorial Sloan-Kettering Cancer Center) presented the structure of a budding yeast Dicer protein from Saccharomyces castellii. This ‘Dicer’ lacks a
PAZ domain, which normally allows Dicer to recognize
the two-nucleotide 3’ overhang of small RNA precursors,and contains only a single RNase III domain. The© 2010 Ameres and Fukunaga; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Ameres and Fukunaga Silence 2010, 1:8
http://www.silencejournal.com/content/1/1/8

structure revealed that the protein dimerizes to generate
two catalytically active centers, with each having thepotential to cleave one strand of the dsRNA, leaving
duplex ends with overhangs two nucleotides in length.
The uniform product length (23 nucleotides) observed invitro, may reflect dimer-dimer interactions on duplex
RNA, a novel strategy to assure a uniform length of Dicer
products in a small RNA pathway.
A hitherto underappreciated feature of Dicer proteinsin higher eukaryotes is the helicase domain, the functionof which is not understood. Brenda Bass (University ofUtah) showed that in Caenorhabditis elegans the helicase
domain is dispensable for pre-miRNA processing; expression of a helicase mutant Dicer in Dicer null mutant animals rescued the phenotype caused by loss of miRNAs.
However, deep sequencing revealed that Dicer helicase
mutants lose primary endo-siRNAs. In vitro processing
experiments suggest that the structure of endo-siRNA
precursor dsRNAs versus the structure of pre-miRNAs
might be the discriminating factor, because dsRNA bearing a blunt end or a 5’ overhang could not be processedby a Dicer lacking a functional helicase domain.
The initial step in miRNA biogenesis, primary (pri)miRNA processing, can be modulated by accessory proteins such as SMAD proteins, as reported by Brandi
Davis (Tufts University) [6]. Davis described experiments that suggest that SMAD proteins associate with
p68, Drosha and DGCR8 on the pri-miRNA to promote
conversion of the pri-miRNA into a pre-miRNA. The
recognition step might rely on a conserved sequence inthe center of the designated mature miRNA.
In the second keynote address, James Carrington(Oregon State University) talked about the mechanismsthat produce trans-acting siRNA (tasi-RNA) in plants[7-9]. The association of miR-173 with Ago1 and miR390 with Ago7 is required to generate precise ends on
TAS1 or TAS3 loci transcripts, respectively, and are
required for the recruitment of an RNA-dependent
RNA polymerase to generate dsRNA that is then further
processed into tasi-RNAs. Besides the importance of the
5’ nucleotide of a miRNA in determining its association
with particular Argonaute proteins, the length of miRNAs also seems to be important for tasi-RNA formation:
miRNAs involved in generation of tasi-RNAs are predominantly 22 nucleotides long, whereas plant miRNAs ingeneral are mostly 21 nucleotides long. Interestingly,
shortening miR-173 from 22 to 21 nucleotides interferedwith the generation of tasi-RNAs from the TAS1 locus,
although neither its loading into Ago1 nor its ability tocleave the TAS1 transcript was altered.
miRNA function

A series of talks described new insights into themechanism of small RNA function in various organisms.

Page 2 of 6

David Bartel (Massachusetts Institute of Technology)
presented compelling genome-wide support for the
recent idea that much of the repression mediated bymiRNAs reflects mRNA destabilization rather thantranslational repression [10,11]. He described ribosomeprofiling experiments [12] in the presence or absence ofspecific miRNAs. Changes in ribosome-protected fragments mostly reflected changes in mRNA levels,
whereas there was only a small, although detectable,change in ribosome density on miRNA-regulated transcripts. This was much of a surprise to most in theaudience, because translational repression has long been
considered to be the major mechanism of miRNA function. A lively and extended question and answer session
followed the talk!
Phillip Zamore (University of Massachusetts Medical
School) speculated on the puzzling discrepancy between
the nature of miRNA targets in plants and animals. In
plants, miRNAs usually pair with high or perfect complementarity to their targets, whereas in animals pairing
is usually limited and is often restricted to only the‘seed region’ (nucleotides 2 to 8) of the miRNA guide.
He showed that in Drosophila, extensive complementarity between miRNAs and their target RNAs leads to tailing and degradation of miRNAs. Interestingly, flies havea small RNA protection system that is based on the
methyltransferase Hen1, and methylates small RNAs
associated with Ago2, the effector protein in the RNAi
pathway. The methylation of small RNAs at their 3’
terminal ribose counteracts tailing and degradation, andis required to ensure the integrity and abundance ofendogenous small RNAs in Drosophila. In plants, allmiRNAs are methylated, which might have allowed
high-complementarity targets to evolve. Interestingly,
the target-dependent small RNA tailing and degradation
machinery seems to be conserved in humans.
Tudor Fulga (Harvard Medical School) introduced apowerful tool to study miRNA function in flies [13].
The expression of miRNA sponges (originally established by Phil Sharp’s group in mammalian cell lines[14]) allows uncovering of miRNA null mutant phenotypes with spatiotemporal resolution in transgenic flies
employing the Gal4-UAS system. Although it is still
unclear if miRNA sponges work via competition ormiRNA degradation, this system will be a valuable tool
to understand miRNA function in model organisms
such as Drosophila melanogaster.
Victor Ambros (University of Massachusetts Medical
School) gave an update on how the tripartite motif(TRIM)-NHL protein, NHL-2, functions as a cofactor
for miRNA-induced silencing by enhancing post-transcriptional repression of target mRNAs [15]. NHL-2
associates with CGH-1 (a P-body component) and the
Argonaute proteins, ALG-1 and ALG-2, and theseAmeres and Fukunaga Silence 2010, 1:8
http://www.silencejournal.com/content/1/1/8

interactions are ATP-dependent. Dr Ambros also
reported novel alg-1 mutants identified in a screen forgenetic suppressors of lin-28. These alg-1 mutations
affect amino acids in or surrounding the PIWI domain
and act as antimorphic mutations because they have amore detrimental effect on the organism than does anull mutant. Such an effect might be explained by apossible competition of mutant Alg-1 with Alg-2, whichin an alg-1 null mutant can take over miRNA
regulation.
Martijn Kedde (Netherlands Cancer Institute)described a switch in RNA structure in the 3’ untranslated region (UTR) of p27 that regulates miRNA accessto its binding site. He suggested that the RNA-binding
protein, Pumilio, binds to the p27 3’ UTR in cycling
cells, opening a hairpin structure and thereby increasing
the accessibility of miRNA binding sites to miR-221 andmiR-222. Growth factor stimulation changes the phosphorylation status of Pumilio, which modifies its affinity
for the p27 3’ UTR binding sites. This might contributeto the upregulation of p27 in quiescent cells, providing
the basis for a novel RNA-binding protein-induced
switch that modulates miRNA-mediated gene regulation.
In the first part of his talk, Dinshaw Patel presented
structural insights into the ternary interaction and structural rearrangements associated with the Thermus thermophilus Argonaute protein, its associated guide DNA,and a target RNA [16]. He also gave an outlook on anovel structure of the bacterial Argonaute protein
bound to an uncleaved DNA target, which allowed
investigation of rearrangements at the catalytic center ofthe wild-type protein. He found that upon formation of
15 perfect base pairs between the DNA guide and its
target, Argonaute undergoes conformational transitions
that lock it into a catalytically competent state, positioning a divalent cation near the scissile phosphate.
Eliza Izaurralde (Max Planck Institute for Developmental Biology) reported that the interaction between
PABC1 and the silencing domain of GW182 results incompetition for eIF4G binding to the 5’ cap, ultimatelyinterfering with mRNA circularization [17]. This
increases the accessibility of the mRNA 3’ end to deadenylating enzymes, contributing to miRNA-mediateddestabilization of mRNAs.
Gene Yeo (University of California, San Diego) discussed the recent identification of endogenous Argonaute binding sites in C. elegans using cross linking,
ALG1 immunoprecipitation, and high-throughput
sequencing (HITS) (with crosslinking immunoprecipitation (HITS-CLIP or CLIP-Seq)) [18]. Yeo and his collaborators are currently trying to identify Argonautebinding sites in human stem cells, which exhibit miRNA
expression profiles distinct from common human celllines.

Page 3 of 6

Biology of small RNAs

Several talks at the meeting reiterated the multifarious
character of miRNAs, being involved in the regulationof a plethora of biological processes. In the second partof her keynote lecture, Narry Kim reported that miR-8/
miR-200 regulate body weight homeostasis in flies andmice [19]. miR-8 in flies and miR-200 in humans target
USH (Usher syndrome)/FOG2 (friend of Gata 2), which
in turn inhibits phosphoinositide 3-kinase (PI3K). Lossof those miRNAs therefore activates fat cell growththrough increased PI3K activity. Their study identified
two novel regulators of insulin signaling, USH/FOG2
and miR-8/miR-200.
Craig Mello (University of Massachusetts Medical
School) presented an historic overview of small RNA
pathways in C. elegans, emphasizing a class of endo-siRNAs called 22G-RNAs. Several distinct 22G-RNA systems act in worms. In the germline, one system requires
worm-specific AGOs, including WAGO-1, an Argonautethat localizes to germ line nuage structures and silences
certain genes, transposons, pseudogenes and cryptic loci[20]. In association with the Argonaute CSR-1, 22GRNAs localize to chromosomes and are required forproper chromosome segregation [21].
Phil Sharp (Massachusetts Institute of Technology)
talked about the role of miRNAs in mammalian cellrobustness. He showed that although misexpression of
miRNAs was repeatedly found to be associated with certain types of cancer, miRNA regulation is not essential
for cancer. A tumor cell line that is defective for
miRNA synthesis can still form tumors. In a reporter
cell line, Dr Sharp tested the threshold for miRNAmediated repression under increased transcription rates
and found that at low levels of reporter expression,
miRNA-mediated repression is strongest, and that highexpression almost completely relieves this type of regulation. Finally, he showed that during stress, the cytoplasmic subcellular localization of mRNAs and miRNAs
is dependent upon formation of poly(ADP-ribose) andthat miRNA repression is reduced under these
conditions.
Deepak Srivastava (Gladstone Institute of Cardiovascular Disease) talked about miRNA regulation of cardiovascular cell fate. He elaborated on individual miRNAs
that govern differentiation and development of embryonicstem cell-derived and induced pluripotent stem cellderived multipotent cardiac progenitors into cardiomyocytes, endothelial and smooth muscle cells, thereby
emphasizing the potential of miRNA manipulation in
influencing the outcome of cell fates and of cellular
homeostasis.
Mark Boldin (Regulus Therapeutics) showed that miR146a is an endotoxin-responsive miRNA, and miR-146
knockout mice are hypersensitive to bacterial challengeAmeres and Fukunaga Silence 2010, 1:8
http://www.silencejournal.com/content/1/1/8

and develop a spontaneous autoimmune-like disorder.
He suggested that miR-146a serves as a molecular brakefor both the inflammatory response and oncogenictransformation of immune cells.
Dasaradhi Palakodeti (University of Connecticut
Health Center) discussed the involvement of small
RNAs in the regenerative capabilities of the planarian
Schmittea mediterrenea. The regeneration of lost tissues
and organs from neoblasts (the equivalents of stem cells
in higher organisms) is sensitive to irradiation, which
depletes the animals of neoblasts. Knockdown of SmedAgo1 by RNA interference (RNAi) leads to drastic
regeneration defects and a decrease in the neoblast
population, implicating the miRNA pathway in regeneration and neoblast function in planaria.
Small RNAs in chromatin formation

Sarah Elgin (Washington University) discussed thediminutive Drosophila melanogaster ’dot’ chromosome,which is mainly occupied by heterochromatin bearing
HP1 and H3K9 dimethylation, causing variegatedexpression of transgenes inserted into diverse regions of
the fourth chromosome. Silencing of reporters inserted
into the 4th chromosome can be suppressed by mutations in aubergine and piwi. HP1 binds to PIWI in vitro,and in the germline, PIWI is needed for HP1 localization at the promoter region of HeT-A transposons.
Shiv Grewal (National Institutes of Health) showedthat in Schizosaccharomyces pombe, the histone methyltransferase Clr4 is required for generation of centromeric siRNAs, partly independent of its role inmethylation of histone H3 lysine 9. He presented evidence suggesting that the Clr4 complex and the RNAi
effector complex, RNA-induced transcriptional silencing(RITS), might be recruited to chromatin in a cooperative
manner. He also showed that loss of either Clr4 or Ago1
in the absence of functional H2A.Z increases the levelsof antisense transcripts, which are usually suppressedwith assistance of Dicer and the exosome. He proposeda model in which heterochromatin and RNAi components cooperate with a variant histone H2A.Z to suppress non-coding and antisense transcripts.
Danesh Moazed (Harvard Medical School) described
Dicer-independent ‘primal small RNAs’ (priRNAs) in S.
pombe [22]. These 22-23-nucleotide RNAs posses a 5’ uracil bias and specifically associate with Argonaute1. priRNAs map to the 3’ UTR of mRNAs and to non-coding
RNAs in centromeric repeat regions, where they mediate
low levels of H3K9 methylation. Convergent transcripts
from centromeric repeats might give rise to antisense
priRNAs, which cause siRNA amplification through theRNA-dependent RNA polymerase complex and Dicer-1.
Stephan Emmerth (Friedrich Miescher Institute)showed that Dicer-1 in the fission yeast S. pombe isPage 4 of 6

localized to the nucleus and is enriched in the nuclear
periphery [23]. Localization requires a short motif,
which constrains nuclear export promoted by thedsRNA binding domain of Dicer-1. Deletion of thismotif results in localization of Dcr1 to the cytoplasm
and causes marked changes in gene expression and failure to assemble heterochromatin.
RNAi and therapeutics

Beverly Davidson (University of Iowa) talked about therapeutic strategies to target Huntington’s disease (HD).
Preliminary in vivo experiments in established mousemodels of this fatal neurodegenerative disorder indicateddisease improvement after silencing both mutant andwild-type huntingtin alleles via RNAi. This could provide an important alternative approach to the as yet
untested single nucleotide polymorphism-based therapy,
which may be useful in ~75% of patients with HD. In
additional work, she showed that dysregulation of miRNAs in brains from patients with HD might contributeto its varying clinical presentations and disease course.
Priti Kumar (Yale School of Medicine) presented progress in strategies for delivering siRNAs into T cells for
antiviral therapy. She described a CD7-specific singlechain antibody conjugated to oligo-9-arginine peptide[24] or lymphocyte function-associated antigen (LFA)-1
integrin antibody conjugated to stabilized nanoparticles[25].
Daniel G. Anderson (Massachusetts Institute of Technology) elaborated on the combinatorial development of
nucleic acid delivery agents using a high throughput
approach to develop biomaterials. He provided updates
on the second generation of siRNA delivery formulations, which allow efficient knockdown in vivo by doses
as low as 0.03 mg/kg in non-human primates [26]. Healso showed preliminary performance tests for simultaneous knockdowns of up to 10 genes.
Sakari Kauppinen (Santaris Pharma) described tiny
locked nucleic acid (LNA) antisense oligonucleotides
targeting the seed of miRNAs to repress their function.
An LNA-anti-miR (SPC3649) targeting miR-122 in theliver has previously been used in mice and non-humanprimate studies to induce a long-lasting decrease inserum cholesterol, and the success story of this LNA
compound continues, because the same strategy has
been introduced as a new anti-hepatitis C virus therapy
in chronically infected chimpanzees [27]. Clinical trials
for SPC3649 are ongoing, and Kauppinen reported thatthe molecule seems to be well tolerated and shows efficacy in healthy human volunteers.
PIWI-interacting (pi)RNAs

Mikiko Siomi (Keio University School of Medicine)described the specific association of a Tudor domain-

Ameres and Fukunaga Silence 2010, 1:8
http://www.silencejournal.com/content/1/1/8

containing protein, Tudor, with Aubergine and Ago3
through symmetric dimethyl-arginine (sDMA) modifications in the PIWI proteins introduced by dPRMT5/Capsuleen/DART5. She suggested that this might beinvolved in the loading of primary PIWI-interacting (pi)RNAs into the effector PIWI proteins [28]. Studies in afly ovarian somatic cell line expressing PIWI but not
Aubergine or Ago3, indicate that PIWI and the RNA
helicase Armitage (Armi) but not Maelstrom (Mael) or
Spindle-E (Spn-E) (both factors implicated in the piRNA
pathway in flies) are required for piRNA accumulation.
Armi co-localizes with the helicase and Tudor domaincontaining protein, Yb, to distinct foci in the cytoplasm
called Yb bodies. Similar colocalization can be observedin fly ovaries and testes. In a model, PIWI and Armi
were proposed to be involved in primary piRNA accumulation in Yb bodies, whereas Mael and Spn-E might
participate in the ‘ping-pong’ amplification loop.
René Ketting (Royal Netherlands Academy of Arts andSciences) presented novel insights into the zebrafish
piRNA pathway. Ziwi and Zili, the two zebrafish PIWI
proteins, localize to nuage, a germline-specific perinuclear structure, and associate with antisense and sense
piRNAs, respectively, exhibiting the characteristic pingpong signature. As in other animals, zebrafish piRNAs
bear a 2’-O-methyl group on their 3’ terminal nucleotide, which protects them from uridylation and destabilization. Interestingly, mutants in the methyltransferase,
Hen1, only develop into males, which are fertile. This is
in contrast to Piwi mutant males, which are sterile, suggesting that in hen1 mutants the piRNA pathway is disturbed rather than disrupted. Again as in flies, the twozebrafish Piwi proteins bear sDMA modifications
required for binding to Tdrd1. Tdrd1 associates with
piRNA precursor transcripts and thus may function in
substrate selection. Furthermore, PIWI proteins are
required for meiosis and cell differentiation in a mannerthat is not related to transposon silencing.
Alex Bortvin (Carnegie Institution of Washington)suggested that an evolutionarily conserved gene, maelstrom, is needed for transposon silencing in the male
mouse germline, and for proper chromosome synapsis
and crossing over in both male and female meiosis [29].

Conclusions
The conference highlighted the expanding roles ofsmall-silencing RNAs in various biological systems, theirfinely tuned biogenesis, regulatory mechanisms andpotential as therapeutics. It is exciting to see that after adecade of intensive study, the field is not running out ofsurprises. In fact, many of the presentations raised captivating questions that left the attendees of the meeting
yearning for next year’s conference.

Page 5 of 6

Acknowledgements
We thank Phil Zamore and Beverly Davidson for organizing the meeting; all
the speakers for magnificent presentations, comments on the manuscript
and for permitting us to discuss their unpublished results; and Zhao Zhang,
Megha Ghildiyal and members of the Zamore lab for comments on the
manuscript. We apologize to those whose presentations could not becovered due to space limitations. SLA is supported by EMBO long term
fellowship (ALTF 522-2008) and an Erwin Schödinger-Auslandsstipendium(Austrian Science Fund FWF, J2832-B09) and RF by JSPS Research
Fellowships for Research Abroad.
Authors’ contributions
SLA and RF wrote, read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 19 February 2010 Accepted: 15 March 2010
Published: 15 March 2010
References
1. Heo I, Joo C, Kim YK, Ha M, Yoon MJ, Cho J, Yeom KH, Han J, Kim VN: TUT4
in concert with Lin28 suppresses microRNA biogenesis through premicroRNA uridylation. Cell 2009, 138:696-708.
2. Heo I, Joo C, Cho J, Ha M, Han J, Kim VN: Lin28 mediates the terminal
uridylation of let-7 precursor MicroRNA. Mol Cell 2008, 32:276-284.
3. Macrae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ, Adams PD,
Doudna JA: Structural basis for double-stranded RNA processing byDicer. Science 2006, 311:195-198.
4. Wang HW, Noland C, Siridechadilok B, Taylor DW, Ma E, Felderer K,
Doudna JA, Nogales E: Structural insights into RNA processing by thehuman RISC-loading complex. Nat Struct Mol Biol 2009, 16:1148-1153.
5. Tomari Y, Matranga C, Haley B, Martinez N, Zamore PD: A protein sensor
for siRNA asymmetry. Science 2004, 306:1377-1380.
6. Davis BN, Hilyard AC, Lagna G, Hata A: SMAD proteins control DROSHAmediated microRNA maturation. Nature 2008, 454:56-61.
7. Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL,
Chapman EJ, Fahlgren N, Allen E, Carrington JC: Specificity of
ARGONAUTE7-miR390 interaction and dual functionality in TAS3 transacting siRNA formation. Cell 2008, 133:128-141.
8. Cuperus JT, Montgomery TA, Fahlgren N, Burke RT, Townsend T,
Sullivan CM, Carrington JC: Identification of MIR390a precursor
processing-defective mutants in Arabidopsis by direct genome
sequencing. Proc Natl Acad Sci USA 2010, 107:466-471.
9. Montgomery TA, Yoo SJ, Fahlgren N, Gilbert SD, Howell MD, Sullivan CM,
Alexander A, Nguyen G, Allen E, Ahn JH, Carrington JC: AGO1-miR173
complex initiates phased siRNA formation in plants. Proc Natl Acad Sci
USA 2008, 105:20055-20062.
10. Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP: The impact of
microRNAs on protein output. Nature 2008, 455:64-71.
11. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N:
Widespread changes in protein synthesis induced by microRNAs. Nature
2008, 455:58-63.
12. Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS: Genome-wide
analysis in vivo of translation with nucleotide resolution using ribosome
profiling. Science 2009, 324:218-223.
13. Loya CM, Lu CS, Van Vactor D, Fulga TA: Transgenic microRNA inhibition
with spatiotemporal specificity in intact organisms. Nat Methods 2009,
6:897-903.
14. Ebert MS, Neilson JR, Sharp PA: MicroRNA sponges: competitive inhibitorsof small RNAs in mammalian cells. Nat Methods 2007, 4:721-726.
15. Hammell CM, Lubin I, Boag PR, Blackwell TK, Ambros V: nhl-2 Modulates
microRNA activity in Caenorhabditis elegans. Cell 2009, 136:926-938.
16. Wang Y, Juranek S, Li H, Sheng G, Wardle GS, Tuschl T, Patel DJ:
Nucleation, propagation and cleavage of target RNAs in Ago silencing
complexes. Nature 2009, 461:754-761.
17. Zekri L, Huntzinger E, Heimstadt S, Izaurralde E: The silencing domain of
GW182 interacts with PABPC1 to promote translational repression anddegradation of microRNA targets and is required for target release. Mol
Cell Biol 2009, 29:6220-6231.

Ameres and Fukunaga Silence 2010, 1:8
http://www.silencejournal.com/content/1/1/8

Page 6 of 6

18. Zisoulis DG, Lovci MT, Wilbert ML, Hutt KR, Liang TY, Pasquinelli AE,
Yeo GW: Comprehensive discovery of endogenous Argonaute bindingsites in Caenorhabditis elegans. Nat Struct Mol Biol 2010, 17:173-179.
19. Hyun S, Lee JH, Jin H, Nam J, Namkoong B, Lee G, Chung J, Kim VN:
Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control
growth by regulating PI3K. Cell 2009, 139:1096-1108.
20. Gu W, Shirayama M, Conte D Jr, Vasale J, Batista PJ, Claycomb JM,
Moresco JJ, Youngman EM, Keys J, Stoltz MJ, Chen CC, Chaves DA, Duan S,
Kasschau KD, Fahlgren N, Yates JR, Mitani S, Carrington JC, Mello CC:
Distinct argonaute-mediated 22G-RNA pathways direct genome
surveillance in the C. elegans germline. Mol Cell 2009, 36:231-244.
21. Claycomb JM, Batista PJ, Pang KM, Gu W, Vasale JJ, van Wolfswinkel JC,
Chaves DA, Shirayama M, Mitani S, Ketting RF, Conte D Jr, Mello CC: The
Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentricchromosome segregation. Cell 2009, 139:123-134.
22. Halic M, Moazed D: Dicer-Independent Primal RNAs Trigger RNAi andHeterochromatin Formation. Cell 2010, 140:102-113.
23. Emmerth S, Schober H, Gaidatzis D, Roloff T, Jacobeit K, Buhler M: Nuclear
Retention of Fission Yeast Dicer Is a Prerequisite for RNAi-Mediated
Heterochromatin Assembly. Dev Cell 2010, 18(1):102-113.
24. Kumar P, Ban HS, Kim SS, Wu H, Pearson T, Greiner DL, Laouar A, Yao J,
Haridas V, Habiro K, Yang YG, Jeong JH, Lee KY, Kim YH, Kim SW, Peipp M,
Fey GH, Manjunath N, Shultz LD, Lee SK, Shankar P: T cell-specific siRNAdelivery suppresses HIV-1 infection in humanized mice. Cell 2008,
134:577-586.
25. Kim SS, Peer D, Kumar P, Subramanya S, Wu H, Asthana D, Habiro K,
Yang YG, Manjunath N, Shimaoka M, Shankar P: RNAi-mediated CCR5
silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT
mice. Mol Ther 2010, 18:370-376.
26. Love KT, Mahon KP, Levins CG, Whitehead KA, Querbes W, Dorkin JR, Qin J,
Cantley W, Qin LL, Racie T, Frank-Kamenetsky M, Yip KN, Alvarez R, Sah DW,
de Fougerolles A, Fitzgerald K, Koteliansky V, Akinc A, Langer R,
Anderson DG: Lipid-like materials for low-dose, in vivo gene silencing.
Proc Natl Acad Sci USA 2010, 107:1864-1869.
27. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME,
Kauppinen S, Orum H: Therapeutic silencing of microRNA-122 in primateswith chronic hepatitis C virus infection. Science 2010, 327:198-201.
28. Nishida KM, Okada TN, Kawamura T, Mituyama T, Kawamura Y, Inagaki S,
Huang H, Chen D, Kodama T, Siomi H, Siomi MC: Functional involvementof Tudor and dPRMT5 in the piRNA processing pathway in Drosophila
germlines. EMBO J 2009, 28:3820-3831.
29. Aravin AA, van dHGW, Castañeda J, Vagin VV, Hannon GJ, Bortvin A:
Cytoplasmic compartmentalization of the fetal piRNA pathway in mice.
PLoS Genet 2009, 5:e1000764.
doi:10.1186/1758-907X-1-8
Cite this article as: Ameres and Fukunaga: Riding in silence: a little
snowboarding, a lot of small RNAs. Silence 2010 1:8.

Submit your next manuscript to BioMed Central
and take full advantage of:• Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit