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As siRNAs derive from such long
double-stranded RNAs, one could
expect siRNA precursors toconstitutively activate the interferon
response. except that the whole
pathway seems to be inactive in mouse
oocytes [15]. Identifying the mouse
tissues devoid of an interferon
response could prove helpful in the
search for endogenous siRNA function.
It can be expected, at least, that one
particular feature of siRNAs will notmake that task easy: endogenous
siRNAs are expressed at very low
levels — much lower than typical
miRNAs. This may be linked tothe activity of siRNAs: as guides
for the endonucleolytic cleavage of
their targets, they act catalytically,
whereas most animal miRNAs
act stoichiometrically (the target
needs to be bound by the miRNA tobe repressed, until it is degraded).
These observations suggest an
impressive, almost scary, possibility.
After the miRNA era (where so many
functions have been ascribed tomiRNAs, in so many physiologicalprocesses), we may be entering the‘siRNA era’. How many biologicalpathways will involve siRNAs? As
siRNAs can act catalytically, minuteamounts of these novel regulators
could have tremendous effects;

undetected small RNAs may lie behind
unexplained phenomena. Clearly, theexploration of small regulatory RNAs is
not over.

9.

10.

References
1. Watanabe, T., Totoki, Y., Toyoda, A.,
Kaneda, M., Kuramochi-Miyagawa, S.,
Obata, Y., Chiba, H., Kohara, Y., Kono, T.,
Nakano, T., et al. (2008). Endogenous siRNAs
from naturally formed dsRNAs regulatetranscripts in mouse oocytes. Nature 453,
539–543.
2. Tam, O.H., Aravin, A.A., Stein, P., Girard, A.,
Murchison, E.P., Cheloufi, S., Hodges, E.,
Anger, M., Sachidanandam, R., Schultz, R.M.,
et al. (2008). Pseudogene-derived smallinterfering RNAs regulate gene expression inmouse oocytes. Nature 453, 534–538.
3. Babiarz, J.E., Ruby, J.G., Wang, Y., Bartel, D.P.,
and Blelloch, R. (2008). Mouse ES cells
express endogenous shRNAs, siRNAs,
and other Microprocessor-independent,
Dicer-dependent small RNAs. Genes Dev. 22,
2773–2785.
4. Lagos-Quintana, M., Rauhut, R., Lendeckel, W.,
and Tuschl, T. (2001). Identification of novel
genes coding for small expressed RNAs.
Science 294, 853–858.
5. Stefani, G., and Slack, F.J. (2008). Small
non-coding RNAs in animal development.
Nat. Rev. Mol. Cell. Biol. 9, 219–230.
6. Bernstein, E., Kim, S.Y., Carmell, M.A.,
Murchison, E.P., Alcorn, H., Li, M.Z., Mills, A.A.,
Elledge, S.J., Anderson, K.V., and Hannon, G.J.
(2003). Dicer is essential for mouse
development. Nat. Genet. 35, 215–217.
7. Suh, N., Baehner, L., Melton, C., Shenoy, A.,
Chen, J., and Blelloch, R. (2010). MicroRNA
function is globally suppressed during mouse
oocyte maturation and preimplantation
development. Curr. Biol. 20, 271–277.
8. Ma, J., Flemr, M., Stein, P., Berninger, P.,
Malik, R., Zavolan, M., Svoboda, P., andSchultz, R.M. (2010). Loss of microRNA

11.

12.

13.

14.

15.

function during oocyte growth in mouse.
Curr. Biol. 20, 265–270.
Murchison, E.P., Stein, P., Xuan, Z., Pan, H.,
Zhang, M.Q., Schultz, R.M., and Hannon, G.J.
(2007). Critical roles for Dicer in the female
germline. Genes Dev. 21, 682–693.
Tang, F., Kaneda, M., O’Carroll, D., Hajkova, P.,
Barton, S.C., Sun, Y.A., Lee, C.,
Tarakhovsky, A., Lao, K., and Surani, M.A.
(2007). Maternal microRNAs are essential formouse zygotic development. Genes Dev. 21,
644–648.
Chu, C.Y., and Rana, T.M. (2006). Translation
repression in human cells by microRNAinduced gene silencing requires RCK/p54.
PLoS Biol. 4, e210.
Eulalio, A., Behm-Ansmant, I., Schweizer, D.,
and Izaurralde, E. (2007). P-body formation is aconsequence, not the cause, of RNA-mediatedgene silencing. Mol. Cell. Biol. 27, 3970–3981.
Claycomb, J.M., Batista, P.J., Pang, K.M.,
Gu, W., Vasale, J.J., van, Wolfswinkel, J.C.,
Chaves, D.A., Shirayama, M., Mitani, S.,
Ketting, R.F., et al. (2009). The Argonaute
CSR-1 and its 22G-RNA cofactors are
required for holocentric chromosome
segregation. Cell 139, 123–134.
Bushati, N., and Cohen, S.M. (2007). microRNA
functions. Annu. Rev. Cell. Dev. Biol. 23,
175–205.
Stein, P., Zeng, F., Pan, H., and Schultz, R.M.
(2005). Absence of non-specific effects of
RNA interference triggered by long
double-stranded RNA in mouse oocytes.
Dev. Biol. 286, 464–471.

Université de Toulouse, UPS, Laboratoire de
Biologie Moléculaire Eucaryote, F-31000,
France, and CNRS, Laboratoire de Biologie
Moléculaire Eucaryote, F-31000 Toulouse,
France.
E-mail: seitz@ibcg.biotoul.fr

DOI: 10.1016/j.cub.2009.12.027

Short RNAs: How Big Is This Iceberg? in Drosophila, human, mouse, rat,
Recent studies have reported the identification of piwi-associated RNAs
(piRNAs) in Drosophila somatic cells. Interestingly, these piRNAs derive fromthe 30 untranslated regions of a subset of transcribed protein-coding genes
and, experimentation suggests, might control the expression of otherprotein-coding transcripts. Studies of additional organisms support the newpathway’s presence across animals.
Isidore Rigoutsos
The RNA revolution is upon us. The
last decade and a half has witnesseda flurry of research activity revolving
around RNA, a molecule that for many
years was thought to play only ancillaryroles in the workings of a cell. Aided bytechnological and scientific advances,
the repertoire of non-coding RNAs, i.e.
RNAs that do not code for proteins, hasbeen expanding steadily [1].
A particular class of short RNAs,
microRNAs, has so far commanded thelion’s share of researchers’ attention.

Approximately 22 nucleotides long,
microRNAs have been shown topost-transcriptionally regulate theirtargets in a sequence-dependent
manner [2]. Originally believed toact through the 30 untranslated region(30 UTR) of the targeted mRNAs,
microRNAs were recently shown totarget the mRNA’s amino-acid coding
sequence (CDS) as well [3,4]. Recently,
piwi-associated RNAs (piRNAs),
another intriguing class of short RNAs
with lengths between 23 and 30
nucleotides, burst onto the stage [5–8].
To date, piRNAs have been reported

Xenopus, zebrafish and the worm,
and their known numbers are in thehundreds of thousands.
Drosophila piRNAs deriving fromheterochromatic loci that are rich innested, truncated, or damaged
repeat elements, termed piRNA‘clusters’, have been among thebetter studied. Such piRNAs
participate in the ‘ping-pong’ cycle,
an auto-amplification loop that isconserved in many metazoans andconstitutes an adaptive immune
response that maintains genomic
integrity by suppressing the expressionof transposons and other repetitive
sequences [9,10].
In addition to piRNAs generated via
the ping-pong cycle, other piRNAs that
do not depend on auto-amplification
have also been reported. The specificsof their generation had remained
elusive for a while, but recent studies
by the laboratories of Mikiko andDispatch
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Gene y

tj

5´
3´

brat

3´
5´

Gene xGeneration of 3´UTRs into piRNAs
mRNAs

Protein x

Protein products

BRAT

Protein y

TJ

3´

5´

3´UTR-piRNAs
3´

piwi

5´

TJ

Piwi

piRNAs loaded
onto Piwi

TJ activates piwi transcription

Piwi

Piwi
Piwi

Piwi

Piwi
Piwi

Piwi

Piwi

Piwi

Piwi

Piwi
Piwi

Piwi

Piwi

Piwi

Piwi

Piwi

Piwi

Piwi
Piwi

Piwi

Piwi

Piwi

Putative target
3´
Piwi
Piw
wPiwi
wi
i PiwifasIII intron

5´
Piwi
Piw
wPiwi
wi
i Piwi

Piwi
Piw
wPiwi
wi
i wiPiwi

Haruhiko Siomi, and Eric Lai, havebroken new ground, revealing
surprising results with potentially very
important ramifications [11–13].
A common element in the work byboth teams is their use of a Drosophila
ovarian somatic cell line (OSS),a homogeneous and stable cellline derived from a parental cell line
(fGS/OSS) comprising a mix of female
germline stem cells and sheets ofsomatic cells. This was a very
important design choice in that it
removed one confounding factor ofprevious piRNA studies, namely theuse of systems comprisingheterogeneous cell types.
A combination of qRT-PCR andWestern blot analysis by the two teams
established the presence of Piwi in
OSS, and the absence of both
Aubergine (Aub) and Argonaute 3(AGO3), the latter two being required
for the production of piRNAs throughthe ping-pong cycle. Immunostainingof OSS revealed Piwi’s nuclear
localization but left open the question
as to whether such localization was
required for the production and loadingof piRNAs. The Siomi team [13]designed and studied three mutants:
a Piwi mutant lacking its nuclear
localization signal expectedly localized
to the cytoplasm but could still loadpiRNAs, just like the wild type;a second Piwi mutant that was
deprived of its Slicing faculty throughmutations in its PIWI domain also
maintained the ability to load piRNAs;and finally, a double mutant that was
Slicer-deficient and also lacked its
nuclear localization signal continued toload piRNAs even when endogenous
Piwi was depleted. These findings
suggest that the process at hand,unlike the ping-pong cycle, does notdepend on Piwi’s Slicing ability andthat piRNA production and loading on
Piwi occur in the cytoplasm. In parallel
work, the Lai team [12] analyzed newly
generated and previously publisheddeep sequencing data and found noevidence of the sequence biases thatare the hallmarks of the ping-pongcycle, namely a preponderance ofuridine in the first piRNA position andadenine in the tenth, and theexistence of piRNA pairs containing
complementary sequences in the firstten positions from their 50 end.
Combined, these results indicatethat the piRNAs present in the OSS
cells are generated independently of
Aub/AGO3 and the auto-amplification

3´

5´
5´

Piw
i
P wi
Piw
Piwi
wPiwi
i PiwiPi iwi iw
Piwi
Pi
Piw
Piw
Pi
Piw
Piwi
wi
Piwi
Piw
P
wii

3´
fasIII

3´

5´

Piw
Piwi
wPiwi
wi
i Piwi
Pi i
Piwi
Pi
Piw
Piw
Piwi
wi
w
Piwi
Piw
P
wii

Piw
Piwi
wPiwi
wi
i Piwi
Pi i
Piwi
Pi
Piw
Piw
Piwi
wi
w
Piwii
P

? ?? ? ?
? Unknown targets
????
??? ?
Current Biology

Figure 1. Summary depiction of the findings described in [11–13].
Among the transcribed genes, a subset (e.g. tj, brat, hypothetical gene y and others) gives rise
to piRNAs through the 30 UTRs of their mRNAs. Abundant transcription does not appear to bea prerequisite, and not every transcribed gene’s 30 UTR will generate piRNAs (e.g., hypotheticalgene x). The generation of these piRNAs that are sense to the source 30 UTR occurs in parallelwith the translation of the mRNA. Among the piRNA-producing genes in the OSS cell line, tj
and brat are the two most prolific ones. Once made, the TJ protein appears to facilitate/initiate
transcription of piwi and the production of Piwi. The generated piRNAs are subsequently
loaded on Piwi in the cytoplasm. Preliminary indirect evidence suggests that the complex
carrying some of the tj-piRNAs may target the primary fasIII transcript within its first intron.
Given the high number of the generated distinct piRNAs, it is reasonable to assume that there
is an abundance of targets that remain to be elucidated. Color convention: blue, 50 UTR; red,
CDS; yellow, 30 UTR; cyan, intronic sequence; gray, intergenic sequence.

loop. But where do these piRNAs
come from?
Mapping of the piRNAs on the
Drosophila genome by both teams
revealed the flamenco locus as oneof the sources [12,13]. In addition to
flamenco, and rather unexpectedly,
the two teams discovered that manyof the sequenced piRNAs were sense
to, and derived from, the 30 UTRs of
Drosophila mRNAs (Figure 1). The
deeper sequencing carried out by Lai’steam [11], as reported in a recent issue
of Current Biology, allowed them toidentify many tens of genes generating
greater than 1,000 piRNAs from their
30 UTRs. In fact, these mRNA-derived
piRNAs were comparable inabundance to microRNAs in OSS. It is
worth emphasizing here that despiteusing different platforms, sequencing
at different depths, and employing

different thresholds, the two teams’
lists of top-ranking mRNAs, vis-à-vis
the piRNAs that the mRNAs
generated, were in agreement. The
highest-ranking mRNA in both lists
corresponded to traffic jam (tj), a geneencoding a Maf-bZIP transcription
factor that is necessary for controlling
gonad morphogenesis in the fruit
fly. Another high-ranking
piRNA-generating mRNA was brat.
Given that tj’s mRNA apparently
serves two purposes, namely protein
synthesis and piRNA production, is themRNA pre-processed in any way prior
to entering the primary piRNA
pathway? The answer appears to be
negative. Indeed, Northern blot
analysis by the Siomi team [13] usingprobes targeting, respectively, tj’s
CDS and 30 UTR revealed a single bandof the same length, suggesting noCurrent Biology Vol 20 No 3
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separation of the primary mRNA into
segments.
Further analysis of data from recent
studies of Drosophila mutants [14,15],and of newly generated data [11] inconjunction with RNAi studies [13],allowed the two teams to establish thatzucchini is involved in the 30 UTR-piRNA
pathway. Also, study of larval ovaries oftj mutants by the Siomi team [13]showed TJ to be required for piwi
activation in Drosophila gonadal
somatic cells. Notably, there areseveral potential Maf binding sites nearpiwi’s putative transcription start site:
for some of them, chromatin
immunoprecipitation experiments
indicated a direct association with TJ(Figure 1).
Once produced and loaded on Piwi,
the tj-derived piRNAs have the ability tosuppress targets. By searching fortargets with (near) complementarity to
tj’s 30 UTR piRNAs, the Siomi team [13]was able to identify fasIII as one suchcandidate target gene. Interestingly, allthree putative sites of interaction withthe sequenced piRNAs were in fasIII
introns (Figure 1). Quantitative RT-PCR
analysis showed that fasIII is
upregulated in piwi- and tj-mutants,
supporting the conjecture that fasIII isa piRNA target.
However, are the above results a
tj-specific or, perhaps, aDrosophila-specific peculiarity?
A wide-ranging investigation by Lai’steam [11] provides the answer. Analysis
of public data and additional primarydata that they generated revealed
strong 30 UTR piRNA production inmouse testis as well. These mouse
piRNAs were proportionally highest in
pre-pachytene testis (10 days
postpartum) wherein Mili was foundto be the predominant carrier of
30 UTR-piRNAs, in agreement withearlier observations [16]. Notably,
despite the difference in the carrier
molecule, the identities of the gene
sources of the Mili- and Miwi-loaded
30 UTR-piRNAs were relatively similar.
Further analysis also allowed Lai’steam to reclassify previously publishedpiRNA clusters as 30 UTR-derived ones.
The team also analyzed piRNAs fromX. tropicalis eggs that associated with
the Y12 monoclonal antibody andidentified numerous 30 UTR-piRNAs
there as well, albeit they were not as
abundant as in the mouse and in
Drosophila. The findings of the Laigroup [11] together with corroborating
results by the Siomi team [13] (obtainedby studying piwi expression in mouse
tj-mutant testis) suggest conservationof the identified pathway acrossanimals.
Do all 30 UTRs give rise to piRNAs?
Interestingly, by studying sequenced
reads in conjunction with gene
expression data in OSS, Lai’s teamshowed that high-expression is nota sufficient condition for the generationof piRNAs. Instead, they foundpreference for mRNA substrates
belonging to select gene ontology (GO)categories such as post-transcriptional
gene regulation, regulation of structure
and organ development,
morphogenesis, cell differentiation,and others. A similar analysis of mouse
testis data revealed analogous GO
biases.
These above results provide
evidence in support of a previously
reported, computationally derived
framework that linked intergenic/
intronic regions with exons throughshort motifs termed pyknons [17].
Pyknons are organism-specific,
variable-length, statistically significant
nucleotide motifs that were arrived atusing purely computational means.
By definition, the pyknons of a given
genome occur multiple times inintergenic and/or intronic regions, aswell as at least once in an exon. Despitea pyknon’s multiple occurrences
in a genome, not all instances of the
motif necessarily coincide withretroelements [17]. They were originallya genomic curiosity [18] but after
piRNAs identified by deep sequencing
became available [5–8], it was apparent
that pyknons had computationallypresaged the existence of piRNAs,
both in terms of length and sequence.
At the same time pyknons predicted
putative regulatory connections
between exonic and non-exonic
sequences, similar to that reportedby the Siomi group [13]. Notably,
several of the GO categories that were
found enriched in the original pyknon
analysis [17] agree with those identified
by Lai’s team [11] as associated with
30 UTR-piRNAs. Subsequent analysis
showed that human and mousepyknons are over-represented in the
introns of genes belonging to specific
GO categories, and that the categories
are the same in the two genomes
despite the absence of conservationof the underlying intronic sequences[19]. Additionally, several examplesof pyknons and published piRNAs
that are antisense to introns havebeen reported [19], underscoring
the connection between pyknonsand piRNAs and in accordance withthe putative fasIII findings by Siomi’steam [13].
The pyknon framework anticipatesthat each mRNA region (50 UTR,
CDS, 30 UTR) is associated(as source, target, or perhaps both)to distinguishable-by-sequencecollections of short RNAs with eachregion being linked to distinct GO
categories: glimpses of this areevident in the results generated by Lai’steam [11]. If we now combine thismultitude of connections witha previous report on the differential
expression of a human pyknon
containing genomic region in a disease
context [20], it becomes increasingly
likely that these findings by the Siomi
and Lai teams [11–13] represent further
evidence that the community may havestumbled upon the tip of a regulatory
iceberg of potentially staggering
proportions.
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Morris, P., Brownstein, M.J.,
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Niki, Y., Berezikov, E., and Lai, E.C. (2009).
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13. Saito, K., Inagaki, S., Mituyama, T.,
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Bioinformatics and pattern Discovery
Group, Computation Biology Center,
IBM Thomas J. Watson Research Center,
PO BOX 218, Yonktown Heights,
NY 10598, USA.
E-mail: rigoutso@us.ibm.com
DOI: 10.1016/j.cub.2009.12.036