Available online at www.sciencedirect.com

RNA silencing in germlines—exquisite collaboration of Argonaute
proteins with small RNAs for germline survival
Mikiko C Siomi1,2 and Satomi Kuramochi-Miyagawa3,4
As the proper development of germlines is vital for species
preservation, elaborative, regulatory systems for gene
expression must operate in germlines. One such system is RNA
silencing, sequence-specific gene silencing mechanisms
mediated by small RNAs of 20–30 nucleotides long. Indeed,
recent studies have revealed that various types of small RNAs
are expressed germline-specifically. To preserve thegermlines, they collaborate with Argonaute proteins, thecatalytic engines in RNA silencing, to inhibit injurious,
parasitic genes, transcriptionally or post-transcriptionally.
This chapter summarizes the exquisite collaboration of
Argonaute proteins with small RNAs in the RNA silencing
mechanisms necessary for germline survival in Drosophila
and mice.
Addresses
1
Keio University School of Medicine, Tokyo 160-8582, Japan
2
Japan Science and Technology Agency (JST), CREST, Saitama 3320012, Japan
3
JST, PREST, Saitama 332-0012, Japan
4
Osaka University Medical School and Graduate School of Frontier
Biosciences, Osaka 565-0871, Japan
Corresponding authors: Siomi, Mikiko C (siomim@sc.itc.keio.ac.jp) andKuramochi-Miyagawa, Satomi (smiya@patho.med.osaka-u.ac.jp)

Current Opinion in Cell Biology 2009, 21:426–434
This review comes from a themed issue on
Nucleus and gene expression
Edited by Elisa Izaurralde and Phillip Zamore
Available online 19th March 20090955-0674/$ – see front matter# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2009.02.003

Introduction
Gene silencing mediated by small RNAs of 20–30
nucleotides (nt) long is collectively called RNA silencing. The key findings that spawned RNA silencing
research, particularly in animals, were the identificationof lin-4 miRNA (micro RNA) as a negative regulator of
expression of a heterochronic gene, lin-14 [1] and thediscovery of RNA interference or RNAi [2]. The highconservation of let-7 among species further accelerated
the field [3]. Later, various kinds of endogenous smallRNAs, not only well-known miRNAs but also newcomers, such as piRNAs (PIWI-interacting RNAs)and esiRNAs (endogenous siRNAs), were identified
in organisms ranging from yeast and algae to humans[4–7].
Current Opinion in Cell Biology 2009, 21:426–434

Small RNAs function in RNA silencing by forming acomplex with the Argonaute proteins. In the complex, theArgonaute proteins and small RNAs play different roles;
small RNAs guide the Argonaute proteins to their targets,
while the Argonaute proteins exert enzymatic activities
for inhibiting gene expression, mostly by cleaving transcripts or by blocking protein synthesis [4–7]. In thisreview, we focus on PIWI-interacting RNAs (piRNAs),
germline-specific small RNAs in Drosophila and mice. As
the name indicates, piRNAs are known to be associatedwith the PIWI proteins, which are germline specific
Argonaute proteins. The main issues focused on in thisreview include the mechanisms of piRNA biogenesis andthe mode of action of RNA silencing mediated by the
PIWI-piRNA complex. A relatively newly identified
small RNA, esiRNA, also appears in this review, because
the outcome of esiRNA function, to some extent, overlaps
with that of piRNA function.

Silencing in Drosophila germlines
Earlier studies showed that the piwi (P element-induced
wimpy testes) gene in Drosophila is an essential factor in
germline stem cell (GSC) self-renewal [8]. Further investigation demonstrated that mutations in piwi cause hyperactivation of retrotransposons and impact on theirmobility [9]. The involvement of piwi in controlling
retrotransposon activity has since become obvious.
Identification of small RNAs associated with Piwi in
ovaries revealed that Piwi is specifically loaded with asubset of small RNAs [10,11], originally termed as
rasiRNAs (repeat-associated small-interfering RNAs) of
24–30 nt long [12]. rasiRNAs were recently renamed aspiRNAs, because the PIWI orthologs in other organisms,
such as mice and fish, also associate with the same sort ofsmall RNAs [13]. piRNAs associated with Piwi in Drosophila ovaries are mainly derived from intergenic repetitive
sequences including transposons in the genome [10,11].
An in vitro assay system showed that Piwi exhibits slicer
activity [10]. These results implied that Piwi may function in silencing through a mechanism analogous to RNAiby cleaving gene transcripts that base-pair with a piRNAloaded onto Piwi. However, Piwi is localized in the
nucleus [8,10,11]. Furthermore, recent studies haveshown that Piwi forms a complex with HP1, Heterochromatin protein 1, which is involved in heterochromatinization [14]. Thus, Piwi may also be involved in genesilencing at the transcriptional level. Intriguingly, thePiwi-HP1 complex was shown to induce gene ‘activation’rather than ‘silencing’ [14]. In what circumstance Piwi
triggers gene activation and what mechanisms control thebidirectionality of Piwi function still remain elusive.
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RNA silencing in germlines—exquisite collaboration of Argonaute proteins Siomi and Kuramochi-Miyagawa 427

Another member of the PIWI family, aubergine (aub), wasshown to be necessary for pole cell formation [15] and has
an impact on the mobility of transposons [16]. Later, it
was revealed that Aub in ovaries is associated with piRNAs [11,17,18], as is Piwi [10,11]; the link between
Aub and transposon silencing was thus strengthened.
Small RNAs associated with Aub in testes were also
examined. Needle-shaped aggregation of the Stellate
protein in testes causes spermatogenic defects [19].
Genetic studies revealed the involvement of su(ste) (suppressor of stellate) in stellate silencing. Non-coding su(ste)shows a strong homology to stellate at the nucleotide
sequence level and is located as repetitive genes on
the Y chromosome. However, the molecular mechanismof stellate silencing remained obscure. Later, biochemical
analyses showed that piRNAs originating from the su(ste)repeats are specifically loaded onto Aub and that the AubpiRNA complex immunoisolated from testes is able tocleave the stellate transcript [18,20]. A direct link between
stellate silencing and su(ste) repeats was uncovered(Figure 1). piRNAs associated with Aub in testes also
showed strong complementarities to vasa, a factor necessary for germline development [18]. Indeed, in aub mutant

testes, VASA expression is upregulated. These findings
demonstrated that piRNAs might also function in silencing protein-coding genes.
Sequencing analysis of piRNAs in ovaries revealed thatpiRNAs loaded onto Piwi and Aub are mainly derived
from the antisense transcripts of retrotransposons,
whereas AGO3, the third and the last member of the
PIWI family, preferably binds piRNAs from the sense
strand. Moreover, Piwi-piRNAs and Aub-piRNAs show
a strong preference for uracil at the 50 ends (1st-U),
while AGO3-piRNAs show a bias for adenine at the 10th
nucleotide from the 50 ends (10th-A) [11,17]. Drosophila piRNAs are produced in a Dicer-independent
manner [20] and all the PIWI proteins showed slicer
activity [10,17,18]. Supported by these data, a piRNAbiogenesis model, the amplification loop pathway (a.k.a.
the Ping-Pong pathway) was proposed [11,17](Figure 2). Other genes, including Armitage, SpindleE, Maelstrom (Mael), Krimper, Squash, and Zucchini, mightalso be involved in piRNA biogenesis [20–22]. However, their functions in piRNA biogenesis still remain
unclear.

Figure 1

Aub function in Stellate silencing in Drosophila testes. (a) A model for Stellate silencing mediated by Aub associated with Su(Ste)-originating piRNAs.
Factors necessary for Su(Ste)-originating piRNAs remains unknown. (b) Immunofluorescent image of Drosophila wild-type testis stained with anti-Aub[17]. The white asterisk shows where hub, a somatic cell cluster functioning as niche, is located. (c) Immunofluorescent (anti-Stellate) and DIC imagesof aub mutant testis. Needle-shaped aggregation of the Stellate protein is indicated by red arrowheads.
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Current Opinion in Cell Biology 2009, 21:426–434

428 Nucleus and gene expression

Figure 2

Biogenesis pathways for piRNAs and esiRNAs in Drosophila. The primary antisense transcripts of transposons and piRNA loci (shown in black) areprocessed to piRNAs by unknown mechanisms (called the primary processing), which are loaded onto Aub and Piwi. Then, Aub and Piwi, together with
AGO3, consist a piRNA production system, the amplification loop, by utilizing their slicer activity to determine the 50 end of piRNAs. It is noted that
AGO3 is predominantly associated with sense piRNAs (shown in light gray). The resultant RNP complexes of the PIWI family with piRNAs function insilencing transposons. piRNAs originating from a locus on the X chromosome, flam, are specifically loaded onto Piwi, meaning that flam-piRNAs are
only produced by the primary processing pathway. esiRNAs are processed from transcripts of transposons and esiRNA loci. Dicer2 is involved in
esiRNA biogenesis. Loqs seems to be also involved in the esiRNA processing, but only when long hairpin-shaped transcripts serve as the substrates.
After maturation, esiRNAs are specifically loaded onto AGO2. It is noted that esiRNAs loaded onto AGO2 do not show a strand bias in most cases.

Determination of piRNA origins revealed that a particularlocus on the X chromosome, flamenco ( flam), is a hotspot ofpiRNA production, or is a piRNA cluster [11]. flam wasoriginally identified as a suppressor of specific retrotransposons, gypsy, ZAM, and Idefix [23]. Interestingly, piRNAs originating from flam ( flam-piRNAs) are exclusively
loaded onto Piwi [11]. If flam-piRNAs arise by the
amplification loop system, flam-piRNAs in opposite direction should be detected as AGO3-binders. However, such
piRNAs were not detected. Thus another system forpiRNA biogenesis, distinct from the amplification loop
pathway, must operate [11]. Currently, this system iscalled the primary processing pathway [5] (Figure 2). Piwi
is expressed in ovarian somas, from which Aub and AGO3
are mostly absent [10,11,17,18,24]. It is most likely thatin ovarian somas, only the primary processing systemproduces piRNAs.
Recently, it was shown that piRNAs serve as vectors for
epigenetic information in Drosophila [25]. This finding
is related to a phenotype called ‘hybrid dysgenesis’. The
Current Opinion in Cell Biology 2009, 21:426–434

phenotype, accompanied by progeny sterility, is observed
when fly strains containing a different set of transposons
are crossed. Interestingly, progeny sterility appears only
when male-specific transposons are inherited. Why arefemale-specific transposons tolerated? Recently, Brennecke et al. showed that it might be explained by piRNA
inheritance [25] (Figure 3). In their study, the populations of piRNAs in mothers and daughters in normal anddysgenic crosses were extensively examined, which
revealed that piRNAs are maternally inherited and that
their contents persist from fertilization to adulthood.
Maternal piRNAs were thought to be associated with
the PIWI proteins because their expression, at least of
Aub and Piwi, persists from oocytes to embryos [25](Figure 3). Even in testes, piRNAs are produced andassociated with the PIWI proteins; however, it seems thatthe complexes are not inherited and that this lack ofpaternal deposition is apparently the main cause of hybrid
dysgenesis. It should be noted that the age of the mother
and/or environmental temperature affects progeny sterility in a dysgenic cross [25]. Thus lack of paternalwww.sciencedirect.com

RNA silencing in germlines—exquisite collaboration of Argonaute proteins Siomi and Kuramochi-Miyagawa 429

Figure 3

piRNAs serve as vectors for epigenetic information. A phenomenon, called hybrid dysgenesis, is observed when fly strains containing a different set oftransposons are crossed. Progeny is fertile when transposons (like I-element) possessed only by a parent are maternally inherited (a). However, whensuch transposons are paternally inherited, progeny shows sterility (b). Lack of paternal deposition of the PIWI-piRNA complexes is apparently the maincause of hybrid dysgenesis. (c) Immunostaining images of Drosophila oocytes, embryos, and larval ovary using anti-Aub [17], which indicate that Aub
expression persists from oocytes to embryos.

piRNA inheritance is not the only cause of hybrid dysgenesis.
Recent studies revealed that AGO2, a ubiquitously
expressed Argonaute, is associated with esiRNAs[26,27,28,29,30]. esiRNAs originate from transposons
and other intergenic repetitive regions in the genome, as
in the case of piRNAs. Some esiRNA sequences may
overlap with those of piRNAs. However, esiRNAs andpiRNAs are clearly distinct; piRNAs are 24–30 nt long,associate with the PIWI proteins, and are specifically
expressed in germlines, whereas esiRNAs are 21 nt,
loaded onto AGO2, and are ubiquitous. Their biogenesis
pathways are also distinct; piRNAs are derived fromsingle-stranded RNA precursors through PIWI-slicer
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activities, whereas esiRNAs are produced from dsRNAs
in a Dicer2-dependent manner. Lack of esiRNA production and accumulation causes de-repression of transposons. Thus it is obvious that esiRNAs function intransposon silencing as do piRNAs. Some esiRNAs werealso shown to be involved in silencing protein-coding
genes.

Silencing in mammalian germlines
In mice, IAP (intercisternal A-particle) and LINE1 (longinterspersed nucleotide element) are representative
LTRs (long terminal repeats) and non-LTR retrotransposons, respectively. IAP and LINE1 retrotransposons
were thought to be maintained in a transcriptionally silent
mode by DNA methylation. Indeed, recent studies have
Current Opinion in Cell Biology 2009, 21:426–434

430 Nucleus and gene expression

shown that this was the case. Germline-specific geneknockout studies indicated that DNA methyltransferase
3 (DNMT3) family plays a central role in the de novo
methylation process [31,34,35]. In fact, it is known thatthe transposable DNA elements that are demethylated inE10.5-E12.5 PGCs (primordial germ cells) are de novo
methylated (i.e. reacquisition of DNA methylation) in
gonocyte (prospermatogonium) in the E15.5-18.5 fetal
testis or in primary oocyte after birth [32]. Two membersof the DNMT3 family, DNMT3a2 and DNMT3L, areexpressed in gonocytes at E14-18 [33] in male germ cells.
Although DNMT3L has no methyltransferase activity, it
forms a complex with DNMT3a and is essential for denovo DNA methylation. Indeed, Dnmt3L-deficient mice
fail to establish global de novo DNA methylation oftransposons in their germ cells (Figure 4). Consequently,
transposon expression is enhanced, and the meiosis fails
in the mutants [34,35]. Thus de novo DNA methylation is

Figure 4

Transposon silencing in mouse spermatogenesis. Upper part: During the spermatogenesis, regulatory regions of the transposable DNA elements aredemethylated in PGCs between E10.5 and 11.5. Subsequently, novel DNA methylation (i.e. de novo methylation) takes place in the gonocytes(prespermatogonia) in the fetal testes around E15.5-E18.5. piRNAs expressed during spermatogenesis can be divided into three groups, which
presumably exhibit differential functions at individual stages. The majority of prenatal piRNAs in fetal gonads correspond to transposons, which implies
their functions in de novo DNA methylation of transposon genes. Pre-pachytene piRNAs include both transposon-derived and non-transposon-derived
piRNAs. Pachytene piRNAs are mostly derived from specific piRNA loci. Prenatal piRNAs are bound with MILI and MIWI2, whereas pre-pachytene
piRNAs are associated only with MILI. Pachytene piRNAs are loaded onto MILI and MIWI. Lower part: Horizontal thick lines show the expression
periods of genes indicated (DNMT3L, MILI, MIWI2, MIWI, MAEL, MVH, and TDRD1) during spermatogenesis. Red crosses show the time points whenloss of individual genes shows phenotypic abnormality. How transposon expression and DNA mathylation are affected when individual genes areabrogated in their mutant mice is summarized on the right-hand side. ND: non-determined.
Current Opinion in Cell Biology 2009, 21:426–434

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RNA silencing in germlines—exquisite collaboration of Argonaute proteins Siomi and Kuramochi-Miyagawa 431

essential for suppressing transposons and for spermatogenesis in mice.
Three Piwi genes are found in the mouse genome: Miwi
(mouse Piwi or Piwil1), Mili (Miwi-like or Piwil2), andMiwi2 (or Piwil4). Although all are expressed in germ
lineage cells, their expression patterns are different
during germ cell differentiation (Figure 4). MILI isexpressed from PGCs at E12.5 to round spermatids[36,54], while MIWI2 is mostly expressed in embryonicgonocytes from E15.5; and its expression is diminishedrapidly after birth [37,38]. MIWI is expressed at thelater stage from pachytene spermatocytes to round spermatids in adult testis [39,40]. Loss of Mili and Miwi2
activates retrotransposon gene expression by impairing denovo DNA methylation and induces meiotic failure at thepachytene stage just like Dnmt3L mutant mice [37,41].
Thus Mili and Miwi2 are essential for de novo DNA
methylation of transposons in spermatogenesis. In thisregard, Drosophila and mouse PIWI proteins are functionally different; because it is unlikely that DNA methylation plays any significant role in gene silencing in flies[42].
As in Drosophila, the PIWI proteins in mice, MIWI, MILI,
and MIWI2, are associated with piRNAs. Their size
distribution differs from one another [37,43,44,45](Figure 4). MIWI is bound with 29–31 nt piRNAs,
whereas MILI binds with 24–28 nt piRNAs. MIWI2
associates with 27–29 nt piRNAs. piRNAs expressed in
adult testes, pachytene piRNAs, are mostly derived from
specific loci in the genome and only 12–17% corresponds
to transposons [44–47]. The functions of pachytene piRNAs in spermatogenesis remain unclear. piRNAs in the
fetal prenatal stage, prenatal piRNAs, and in neonate
stage, prepachytene piRNAs, are mostly derived fromtransposons [37,38,43]. The expression of prenatalpiRNAs is impaired in Mili and Miwi2-deficient fetal
gonocytes, indicating that MILI and MIWI2 are involvedin piRNA biogenesis. Considering the DNA methylation
defect found in Mili and Miwi2 mutant testes, PIWI andpiRNAs probably participate in de novo DNA methylationof transposons.
Sequence analysis of piRNAs associated with MILI andMIWI2 revealed that the piRNA production systems,
both the primary processing and the amplification loop
pathways, also exist in mice. However, there are clear
differences between Drosophila and mice [38]. In Drosophila, primary piRNAs mainly correspond to the antisense strand of transposons. However, in mice, primarypiRNAs are largely processed from mRNAs of active
transposons, resulting in production of sense piRNAs.
In E12.5 PGC, the regulatory regions in transposons aredemethylated. As a result, transposons are expressed, andconcomitantly primary sense piRNAs may be processedfrom their transcripts and loaded onto MILI. In gonowww.sciencedirect.com

cytes, where MIWI2 expression occurs, both MILI andMIWI2 enter the amplification cycle for piRNA production. piRNAs associated with MILI and MIWI2 show
a weak but clear strand bias. MILI prefers to bind sense
piRNAs, whereas MIWI2 binds antisense piRNAs. Thus
it is likely that during the piRNA production, MILI
remains associated with primary sense piRNAs, while
MIWI2 is loaded with secondary antisense piRNAs thatare produced from the amplification loop system. Afterbirth, when MIWI2 is no longer expressed, MILI probably continues to operate the cycle alone during spermatogenesis; because MILI, even after birth, is associatedwith both types of piRNAs, one with 1st-U and the otherwith 10th-A [43].
Nuage, the germline-specific cytoplasmic structure foundin a variety of organisms including mice and Drosophila,
has been proposed to play an important role in transposon
silencing [48]. In early spermatogenesis in mice, nuage isobserved in gonocytes, spermatogonia, and pachytene
spermatocytes as multiple granules [49]. Mael, a component of nuage, was shown to be crucial for transposon
silencing in spermatogenesis [50]. Loss of Mael is associated with de-repression of transposons, accompanied by
reduction of DNA methylation (Figure 4). Interestingly,
MILI and MIWI2 also exist in nuage. Subcellular localization of these proteins is dynamic [38,50], shuttlingfrom nuage to the nucleus. All the mutants of mili, miwi2,and mael, undergo meiotic arrest at the same point inspermatogenesis and are linked to DNA damage of
meiotic chromosomes; strongly suggesting their functional relationship. Indeed, Mael interacts with MILI
and MIWI [51]; whether or not MIWI2 interacts withMael is unclear. Mutations in Mael did not change theexpression level of prepachytene piRNAs [50]. Mael
may function in transposon silencing downstream ofpiRNA biogenesis.
In round spermatids, nuage is usually observed as one or
two cytoplasmic granules close to the nucleus, which arecalled chromatoid bodies (CB). CB are likely to be
involved in RNA storing and processing because CB
contain various proteins related to RNA metabolism.
Besides containing MIWI, MILI, and Mael, CB containgerm cell specific components, such as mouse VASA
homolog/DEAD box polypeptide 4 (MVH/Ddx4), Tudor
domain containing 1/ mouse Tudor repeat 1 (TDRD1/
MTR1), and the kinesin motor protein KIF17b, andcomponents of both the miRNA pathway (such as Argonaute proteins, Dicer and GW182) and the RNA-decay
pathway (such as the decapping enzyme DCP1a) [52].
However, the roles of these components in CB are largely
unknown. In Miwi-deficient testis, spermatogenesis isarrested in round spermatids and CB are disrupted[39,53]; thus, MIWI is important for maintaining CB inround spermatids. In contrast to Mili and Miwi2 mutant
testes, transposons are not accumulated in Miwi mutant
Current Opinion in Cell Biology 2009, 21:426–434

432 Nucleus and gene expression

testis. MIWI and MILI interact with piRNAs andmRNAs in polysomes and also some kinds of RNA–
protein complexes in adult testes; thus the MIWI-piRNAand MILI-piRNA complexes may regulate translation
and/or stability of mRNAs necessary for spermatogenesis[53,55]. A recent study showed that MILI may also
positively regulate translation of target genes in germline
stem cells [54] as in the case of Piwi in Drosophila
germlines [14], although such gene activation mechanisms remain obscure.
esiRNAs are also found in mouse oocyte [56,57]. esiRNAs in mice are mapped to retrotrasposons or othergenomic regions that produce transcripts, including
mRNAs and pseudogene transcripts, which are capableof forming dsRNA structures. Inverted repeat structures,
bidirectional transcription at one locus, and sense andantisense transcripts from different loci are the main
sources of the dsRNA precursors. In Dicer and Ago2deficient oocytes, esiRNA levels are decreased. Expression levels of RLTR10 and MTA retrotransposons andprotein-coding transcripts complementary to esiRNAs are
increased. Thus esiRNAs from naturally occurringdsRNAs regulate both protein-coding transcripts andsome transposons in mouse oocytes. LINE1 expression
was not changed in Dicer, Ago2-deficient oocytes, andMili-deficient oocytes. LINE1 expression may beregulated through the combination of piRNAs and esiRNAs in oocytes by a post-transcriptional gene silencing
mechanism, that is, mRNA degradation, whereas IAP
expression is regulated only through piRNAs associatedwith MILI.

Conclusions
Transposons represent a large portion of the genome (inflies and mice, roughly 30% and 40%, respectively[62,63]) and tend to be more active in germlines. Thisis because many germline-specific genes must be
expressed in gonads and thus the chromosomes need
to be kept in a looser structure for a period during theirdevelopment. In such an environment, transposons havea higher innate chance to transpose, which poses a considerable threat to the host species. To restrain transposons, the germline must have acquired powerful
additional means to suppress their activities; namely,
piRNAs and the PIWI proteins that amplify and associatewith piRNAs. In Drosophila, AGO2 in ovaries is loadedwith esiRNAs and exogenous siRNAs and shows slicer
activity in vitro [26,64]. However, AGO2 in fly ovaries
seems to remain inactive in vivo until oocytes become
almost ready to be fertilized [65]. This may be an alternative reason, at least in Drosophila, for the observation that
the PIWI-piRNA system is equipped in germlines forsilencing invasive transposable elements.

Acknowledgements
We thank Haruhiko Siomi for useful discussions. We also thank Y
Kawamura and S Inagaki for providing immunostaining images of Drosophila
testes, ovaries, and embryos. MCS is supported by a grant from NEDO(New Energy and Industrial Technology Development Organization) andby CREST from JST. SKM is supported by PREST from JST. MCS is an
Associate Professor of Global COE for Human Metabolomics Systems
Biology by MEXT.

References and recommended reading
Papers of particular interest published within the period of review havebeen highlighted as:
 of special interest
 of outstanding interest

Silencing in germline: other species
Silencing in the germline is not restricted in Drosophila
and mice. Analogous pathways are present in otherspecies, including fish and nematodes. Zebrafish
expresses two PIWIs, Ziwi and Zili [5,6]. In zebrafish,piRNAs are expressed in both ovaries and testes, as in
Drosophila [58]. Identification and characterization ofzebrafish piRNAs revealed that they are mainly derived
from retrotransposons. Further data suggested that theamplification loop system also exists in zebrafish as well.
Both Ziwi and Zili were shown to be necessary for
maintaining germlines [58,59].
Deep sequencing analysis in C. elegans revealed that thisorganism expresses small RNAs of 21 nt having 1st-U;
they were termed as 21U-RNAs [60]. Their size is very
close to that of esiRNAs; however, 21U-RNAs are
required for maintaining the gemlines, like piRNAs.
The protein partner of 21U-RNAs is PRG-1 (Piwi-related
gene-1), a member of the PIWI family in C. elegans [61].
Accordingly, 21U-RNAs are thought to be C. eleganspiRNAs. They fail to exhibit complementarities to otherexpressed sequences [60,61]; thus their function remains
unclear.
Current Opinion in Cell Biology 2009, 21:426–434

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