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How does the Royal Family of Tudor rule the PIWI-interacting RNA
pathway?
Mikiko C. Siomi, Taro Mannen and Haruhiko Siomi
Genes Dev. 2010 24: 636-646
Access the most recent version at doi:10.1101/gad.1899210

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REVIEW

How does the Royal Family of Tudor rule
the PIWI-interacting RNA pathway?
Mikiko C. Siomi,1,2,3 Taro Mannen,1 and Haruhiko Siomi1,3
1
Keio University School of Medicine, Tokyo 160-8582, Japan; 2Japanese Science and Technology Agency, Core Research for
Evolutional Science and Technology, Saitama 332-0012, JapanPIWI (P-element-induced wimpy testis) proteins area subset of the Argonaute proteins and are expressed
predominantly in the germlines of a variety of organisms,
including Drosophila and mammals. PIWI proteins associate specifically with PIWI-interacting RNAs (piRNAs),
small RNAs that are also expressed predominantly ingermlines, and silence transposable DNA elements andother genes showing complementarities to the sequencesof associated piRNAs. This mechanism helps to maintain the integrity of the genome and the development ofgametes. PIWI proteins have been shown recently tocontain symmetrical dimethyl arginines (sDMAs), andthis modification is mediated by the methyltransferase
PRMT5 (also known as Dart5 or Capsuleen). It was thendemonstrated that multiple members of the Tudor (Tud)family of proteins, which are necessary for gametogenesis in both flies and mice, associate with PIWI proteinsspecifically through sDMAs in various but particular
combinations. Although Tud domains in Tud family
members are known to be sDMA-binding modules,
involvement of the Tudor family at the molecular levelin the piRNA pathway has only recently come into focus.
Argonaute (AGO) proteins associate with small noncoding RNAs of 20–30 nucleotides (nt) to negatively regulate
the expression of genes targeted by the Argonaute–small
RNA complexes (Siomi and Siomi 2009). In this mechanism, termed RNA silencing, genes silenced by thecatalytic activities of Argonaute proteins are involved infundamental cellular processes, such as development,
differentiation, metabolism, and apoptosis (Kim et al.
2009). Thus, Argonaute proteins are essential for many,
if not all, living organisms (Bartel 2009; Malone andHannon 2009; Voinnet 2009).
The number of Argonaute family members in a species
differs; for example, Schizosaccharomyces pombe has
only one Argonaute, while Drosophila and humans
possess five and eight members, respectively (Hutvagner[Keywords: PIWI; PRMT5; RNA silencing; Tudor; piRNA; sDMA]
3
Corresponding authors.
E-MAIL siomim@sc.itc.keio.ac.jp; FAX 81-3-53633266.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1899210.
Freely available online through the Genes & Development Open Access
option.

636

and Simard 2008). Each Argonaute member falls into one
of two subgroups: the AGO and PIWI (P-element-induced
wimpy testis) subfamilies (Farazi et al. 2008). Expressionof AGO members is ubiquitous, whereas PIWI proteinsare detected predominantly in germline cells (Farazi et al.
2008). Depletion of AGO functions often causes developmental defects; for example, Ago2-null mice show embryonic lethality (Liu et al. 2004), while PIWI mutants
show defects in gametogenesis, but otherwise develop
normally (Cox et al. 1998; Harris and Macdonald 2001;
Deng and Lin 2002; Kuramochi-Miyagawa et al. 2004;
Carmell et al. 2007; Li et al. 2009).
AGO proteins associate with microRNAs (miRNAs),
ubiquitously expressed small RNAs that function in RNA
silencing (Kim et al. 2009). In Drosophila, endogenous
siRNAs (endo-siRNAs) are also ubiquitous, and associatewith AGO proteins (predominantly with AGO2). Furthermore, exo-siRNAs (siRNAs exogenously introduced into
cells to artificially induce RNAi) also associate with AGO
in Drosophila and mice (Siomi and Siomi 2009). PIWI
proteins associate specifically with PIWI-interacting
RNAs (piRNAs) in germline cells, although endo-siRNAs
and/or miRNAs are coexpressed with piRNAs in thesecells (Kim et al. 2009). Thus, loading of different kinds ofsmall RNAs onto individual Argonaute proteins is considered to be a ‘‘molecule-specific’’ event.
piRNAs have been studied extensively with regard totheir biogenesis, characteristics, and functions, especially in
Drosophila, fish, nematodes, and mice (in Caenorhabditis
elegans, piRNAs are known as 21U RNAs) (Aravin et al.
2006; Girard et al. 2006; Grivna et al. 2006; Ruby et al. 2006;
Saito et al. 2006; Vagin et al. 2006; Watanabe et al. 2006;
Brennecke et al. 2007; Gunawardane et al. 2007; Houwing
et al. 2007; Batista et al. 2008; Das et al. 2008). piRNAs
are longer than miRNAs and endo-siRNAs by several bases;
for example, in Drosophila, piRNAs range between 24 and30 nt, while miRNAs and endo-siRNAs are ;20–23 nt long.
In addition, piRNAs contain 29-O-methyl groups at their
39 ends, unlike miRNAs (except in plants) (Horwich et al.
2007; Houwing et al. 2007; Kirino and Mourelatos 2007;
Ohara et al. 2007; Saito et al. 2007). piRNAs are derived
mostly from repetitive intergenic DNA elements, including transposons, and these loci are collectively called‘‘piRNA clusters’’ (Aravin et al. 2007a). Protein-coding genes
such as traffic jam (tj) could also account for piRNA

GENES & DEVELOPMENT 24:636–646 Ó 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org

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Tudor in the piRNA pathway

production (Robine et al. 2009; Saito et al. 2009). Because of
these features, and considering their limited expression in
germlines and their specific associations with PIWI proteins, piRNAs are considered to be a unique set of endogenous small RNAs.
Loss of PIWI proteins in Drosophila and mice causes
derepression of transposons and results in severe defects
in gametogenesis (Vagin et al. 2006; Carmell et al. 2007;
Kuramochi-Miyagawa et al. 2008; Li et al. 2009). As
a result, the homozygous mutant lines cannot be maintained. Deletion of a Drosophila piRNA cluster, flamenco(flam), located on the X chromosome, also causes derepression of particular transposons, such as gypsy, ZAM,
and Idefix (Prud’homme et al. 1995; Desset et al. 2003;
Mével-Ninio et al. 2007), because flam gives rise topiRNAs that show strong complementarities to transcripts from these transposons (Brennecke et al. 2007).
These studies have made it very clear that both PIWI
proteins and piRNAs are required for transposon silencing. Targets of PIWI–piRNA complexes are not limited totransposons. In fact, a subset of piRNAs in Drosophila has
been shown to function in silencing protein-coding genes.
The best examples are piRNAs derived from suppressor ofstellate [su(ste)] and tj, which down-regulate proteincoding stellate (ste) and fasciclin III (fas III) genes,
respectively (Livak 1984; Aravin et al. 2001, 2004; Vagin
et al. 2006; Nishida et al. 2007; Saito et al. 2009).
In Drosophila, AGO3 (Argonaute3), Piwi , and Aub(Aubergine) belong to the PIWI protein family, while inmice, MILI, MIWI, and MIWI2 belong to the mouse PIWI
family (Siomi and Siomi 2009). Bioinformatic analyses ofpiRNAs that associate with PIWI proteins in both Drosophila and mice germlines have led to two models for
piRNA biogenesis: the amplification loop pathway (alsotermed the Ping-Pong pathway) and the primary processing pathway (Aravin et al. 2007a; Siomi and Siomi 2009).
Observations in Drosophila support the concepts that, inthe amplification loop pathway, Aub (mainly associatedwith antisense piRNAs, which show a preference fora uracil [U] at the 59 end) and AGO3 (mainly associatedwith sense piRNAs, which show a preference for an
adenine [A] at position 10) reciprocally cleave target
RNAs in sense and antisense orientations, respectively,
and that this reciprocal target RNA cleavage by Aub and
AGO3 constantly gives rise to abundant piRNAs in
germline cells (Brennecke et al. 2007; Gunawardane
et al. 2007). The vast majority of their RNA targets aretransposon transcripts; thus, transposon silencing occurs
in germlines in parallel with piRNA production throughthe amplification loop. In mice, of the three PIWI proteins, MILI and MIWI2 function in the amplification loop
system (Aravin et al. 2007b).
Involvement of Aub and AGO3 in the primary processing pathway, however, is unlikely—at least in somatic
cells of Drosophila ovaries. Evidence supporting thisnotion was obtained from studies on ovarian somatic
cells (OSCs) and ovarian somatic sheets (OSSs). In Drosophila OSCs/OSSs, levels of Aub and AGO3 are below
detection, but piRNAs are abundantly expressed andloaded onto Piwi (Lau et al. 2009; Saito et al. 2009). Deep

sequencing analyses of small RNAs in piRNA-related
mutants also provided strong evidence for an Aub–
AGO3-independent piRNA pathway in somatic cells in
ovaries (Li et al. 2009; Malone et al. 2009). The requirement for MILI and MIWI2 in the mouse somatic primaryprocessing pathway remains undetermined. Factors necessary for primary piRNA processing may include
Zucchini (Zuc), a putative cytoplasmic nuclease, because
loss of Zuc function caused a severe reduction in the expression levels of primary piRNAs, such as flam-originating piRNAs (in ovaries) (Malone et al. 2009) and tj-originating piRNAs (in OSCs) (Saito et al. 2009). dicer mutant
ovaries accumulate piRNAs, similarly to wild-type ovaries (Vagin et al. 2006). Dicer-independent piRNA production was also observed in zebrafish (Houwing et al.
2007). Thus, it seems that both the amplification loop andprimary processing pathways for piRNA production
do not require Dicer. Other genes—such as Armitage,
Spindle E (Spn-E), Maelstrom, Krimper, Vasa, andSquash—might be involved in piRNA biogenesis (Vagin
et al. 2006; Lim and Kai 2007; Pane et al. 2007). However,
the molecular details of the requirement of these genes inpiRNA biogenesis remain unclear.
A recent study has shown that PIWI proteins in
Drosophila, mice, and Xenopus contain sDMAs (symmetrical dimethyl arginines), and that the factor mediating this post-translational modification is PRMT5 (Kirino
et al. 2009). sDMA is one of various methyl group modifications found on specific arginines in protein molecules(Bedford and Clarke 2009). sDMA is known to modify theability of a protein to perform its biological activities. For
example, Sm proteins, factors needed for splicing machinery, contain sDMAs in their arginine–glycine-rich (RG)
domains and associate, through their sDMAs, with a Tud
domain-containing protein, SMN (Survival motor neuron)protein (Brahms et al. 2001; Friesen et al. 2001). This Sm–
SMN association recruits U snRNA, leading to theefficient assembly of U snRNP (Meister et al. 2002). This
whole system serves as a ‘‘gatekeeper’’ that prevents themisassembly of Sm proteins to nontarget RNA and also
prevents Sm protein aggregation (Pellizzoni et al. 2002;
Chari et al. 2008). Other Tud family members interact
with particular proteins through methylated arginines inthe target proteins and to regulate their functions (Côtéand Richard 2005). Genetic studies in Drosophila andmice had indicated that Tud family members are linked
to gametogenesis, even before the discovery of piRNAs
and the characterization of PIWI proteins (Boswell and
Mahowald 1985; Pan et al. 2005; Chuma et al. 2006).
Based on these observations, it was inferred that thebiological functions of PIWI proteins would be regulated
by Tud proteins via specific associations with PIWI-sDMA
modifications. These predictions gradually have beenshown to be correct through recent studies conducted
mainly in Drosophila and mice.
PIWI proteins are sDMA-modified by PRMT5
The first evidence to show that PIWI proteins containsDMAs was provided by Kirino et al. (2009). Kirino et al.

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Siomi et al.

(2009) demonstrated that a protein pool obtained byimmunoprecipitation from mouse testes using an
sDMA-specific antibody, Y12, contained both MILI and
MIWI. PIWI proteins in Drosophila ovaries and Xenopus
oocytes (Xiwi and Xili are the Xenopus PIWI) were also
shown to contain sDMAs by Western blot analyses using
Y12 or SYM11, another sDMA-specific antbody (Kirino
et al. 2009; Nishida et al. 2009). Y12 coimmunoprecipitated piRNAs with PIWI proteins (Kirino et al. 2009),
indicating that sDMA modification does not abolish the
PIWI–piRNA association.
The involvement of PRMT5 in the PIWI-sDMA modification was investigated for two major reasons: (1) Lossof PRMT5 caused complete loss of Sm-sDMA modification in Drosophila (Gonsalvez et al. 2006; Anne et al.
2007), and (2) prmt5 mutants phenocopy aub mutants(Harris and Macdonald 2001). No PIWI-sDMA modification was detectable in prmt5 mutants (Kirino et al. 2009).
Thus, it was concluded that PRMT5 is the factor responsible for the sDMA modification.
The binding capacity of PIWI proteins for piRNAs
seems unaltered by prmt5 mutations (Kirino et al.
2009). However, derepression of a transposon, Het-A,
was observed in prmt5 mutant ovaries (Kirino et al.
2009). This result made it clear for the first time that
prmt5 is tightly connected to transposon silencing.
PRMT5-mediated sDMA modification appears to control
the subcellular localization of PIWI proteins in ovaries
because, in prmt5 mutants, Aub does not concentrate at
the nuage (Nishida et al. 2009), an electron-dense material associated with nurse cell nuclei. This suggests thatnuage localization of Aub is important for silencing.
Whether Aub localization at the posterior pole of oocytes(Kirino et al. 2009), where materials required for primordial germ cell (PGC) specification are accumulated, isaffected by loss of PRMT5 remains controversial (Kirino
et al. 2009; Nishida et al. 2009).
Studies describing mouse prmt5 mutants have not yet
been published. In transgenic mice expressing individual
mouse PIWI proteins, the PRMT5 complex containing
WDR77 (also known as MEP50), a cofactor of PRMT5, wasable to associate with all three PIWI proteins (Vagin et al.
2009). MIWI2 and MILI association with the PRMT5–
WDR77 complex, termed the methylosome, was observedin embryonic testes, while MIWI (and also MILI) association with the methylosome was observed in adult testes.
This was simply due to the stage-specific expression ofindividual PIWI proteins during germline development.
Whether other members of the PRMT family associatewith mouse PIWI proteins remains undetermined.
The Drosophila homolog of WDR77/MEP50 is Valois(Vls) (Anne and Mechler 2005; Cavey et al. 2005). Genetic
disruption of vls results in phenocopies of tud mutants;
both mutants cause a grandchild-less phenotype (Boswell
and Mahowald 1985; Schupbach and Wieschaus 1986).
Vls, like Aub and Tud, is a component of the nuage andpole plasm (Bardsley et al. 1993; Harris and Macdonald
2001). Vls interacts with not only PRMT5, but also Tud,and localization of Tud at the nuage and at the posteriorpole of oocytes depends on Vls expression (Anne and

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GENES & DEVELOPMENT

Mechler 2005). These observations clearly indicate a direct relationship of Vls with Aub and Tud in Drosophila.
sDMA modification status of PIWI proteins in miceand Drosophila
Putative ‘‘sDMA motifs’’ GRG and ARG/GRA (G, glycine; R, arginine; A, alanine), present in animal PIWI
proteins, were suggested by Kirino et al. (2009). The
precise positions of sDMAs in mouse and Drosophila
PIWI proteins were determined later by mass spectrometry (MS). The first R residue identified to have a symmetric dimethyl group was R74 in MILI (Reuter et al.
2009). Interestingly, R74 resides in the sequence FRG (F,phenylalanine), which was not one of the predicted
sDMA motifs. More comprehensive mapping of methylated arginines, including monomethylation, in MILI andMIWI was performed later (Chen et al. 2009; Vagin et al.
2009). Vagin et al. (2009) found that R100, R146, R163,
and R549 in MILI are dimethylated, whereas R95 in MILIand R49 and R371 in MIWI are monomethylated. R74 in
MILI and R14 in MIWI were detected to be both monoand dimethylated. Chen et al. (2009) used endogenous
MILI and MIWI isolated from adult testes and found that
R53 in MIWI can be both mono- and dimethylated. R74,
R83, R95, and R100 in MILI appear to be both mono- anddimethylated, whereas R45, R146, R156, and R163 areonly dimethylated. All dimethylation was most likely
symmetrical (sDMAs). There is some discrepancy between the two analyses that might reflect the methylation status from distinct sources. This may suggest that
the PIWI methylation can be regulated dynamicallythrough spermatogenesis. The current methylation status of mouse PIWI proteins, as determined by MS, issummarized in Figure 1A. The methylation status of
MIWI2 remains undetermined.
In Drosophila, endogenous Aub and AGO3 were subjected to liquid chromatography (LC)-MS/MS analysis.
R11, R13, and R15 in Aub and R4, R68, and R70 in AGO3
were determined to be sDMAs (Fig. 1B; Nishida et al.
2009). R17 in Aub and R72 in AGO3 were predicted to bedimethylated in silico; however, MS as performed by
Nishida et al. (2009) did not reveal these modifications.
Monomethylated Rs were not detected in either Aub or
AGO3 (MC Siomi, unpubl.). The methylation status of
Piwi in ovaries remains undetermined.
Association of members of the Tud familywith PIWI proteins
The tud gene was first discovered in Drosophila as a gene
required for the assembly of the germ plasm, a specialized
cytoplasm containing electron-dense polar granules (Boswell and Mahowald 1985). The tud gene encodes an;280-kDa protein with 11 repeat motifs (Thomson and
Lasko 2004, 2005; Arkov et al. 2006). These domains are
termed Tud domains (Ponting 1997). Tud domains arecommonly found in proteins other than Tud in a wide
range of organisms (Ponting 1997; Talbot et al. 1998;
Maurer-Stroh et al. 2003).

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Tudor in the piRNA pathway

Figure 1. Arginine methylation status of PIWI proteins. Mouse PIWI (A; MILI and MIWI) and fly PIWI (B; Aub and AGO3) N-terminal
sequences are shown with putative sDMA motifs (red). Identified methylation sites (dimethylation [dMe] or monomethylation [mMe])
are shown above the relevant arginine, with the residue numbers below. Underlined sequences indicate the synthetic peptides thathave been used in studies for pull-down assays. The MILI peptide corresponds to L69–T80 and R74 is an sDMA (Reuter et al. 2009). The
MIWI peptide corresponds to M1–T18, and R4, R6, R8, R10, R12, and R14 are sDMAs (Kirino et al. 2010). The Aub peptides correspondto M1–N20 (Kirino et al. 2010) and M1–N25 (Nishida et al. 2009), and R11, R13, and R15 are sDMAs. The AGO3 peptides correspond toM1–K25 and T58–H82, and R4 in the M1–K25 peptide and R68 and R70 in the T58–H82 peptide are sDMAs (Nishida et al. 2009).

Based on data in the NCBI HomoloGene database, miceexpress 26 members of the Tud family (Table 1). Of these,
10 proteins are considered to be members of the Tud
domain-containing (TDRD) family (Table 1). TDRD1,
which contains four Tud domains along with a MYND
(myeloid–nervy–DEAF-1) domain, is necessary for spermatogenesis (Chuma et al. 2006) and interacts with MILI
(Table 2; Reuter et al. 2009; Vagin et al. 2009; Wang et al.
2009). MIWI also interacts with TDRD1 (Table 2; Chenet al. 2009; Kojima et al. 2009; Reuter et al. 2009; Vagin
et al. 2009; Wang et al. 2009), although whether TDRD1shows a tight association with MIWI2 remains controversial (Kojima et al. 2009; Reuter et al. 2009; Vagin et al.
2009; Wang et al. 2009). This type of discrepancy can becaused by different groups using different conditions toanalyze protein–protein interactions.
In vivo interaction assays with various MILI deletionmutants determined a domain necessary for TDRD1interaction (Reuter et al. 2009). This domain was in the
N-terminal region of MILI and contained dimethylated
R74 (Reuter et al. 2009). Treatment with MTA [59-deoxy59-(methylthio)-adenosine], a methyltransferase inhibitor, disrupted MILI-sDMA modification and abolished
the association of MILI with TDRD1 (Reuter et al. 2009;
Vagin et al. 2009). However, unlike full-length MILI, theN-terminal region was not detected by Y12 (Reuter et al.
2009); this implies that sDMA may not be absolutely
necessary for the interaction between MILI and TDRD1.

Loss of the MYND domain in TDRD1 does not affect the
MILI–TDRD1 interaction (Reuter et al. 2009), indicatingthat the association occurs through Tud domains.
Tud domain-containing proteins that can associatewith PIWI proteins in mouse testes are not limited to
TDRD1. Seven Tud domain-containing proteins—TDRD1
to TDRD9, except TDRD3 and TDRD5—were found to be
associated with PIWI proteins by various means, both invivo and in vitro (Table 2; Chen et al. 2009; Kojima et al.
2009; Shoji et al. 2009; Vagin et al. 2009; Vasileva et al. 2009;
Wang et al. 2009; Kirino et al. 2010). sDMA dependency ofsome TDRD proteins to interact with PIWI proteins has
been determined. For example, TDRD6—considered themouse homolog to Drosophila Tud, although TDRD6
contains seven Tud domains (Hosokawa et al. 2007),
whereas Tud contains 11 Tud domains (Thomson and
Lasko 2004, 2005; Arkov et al. 2006)—was able to associatewith MILI and MIWI (Chen et al. 2009; Vagin et al. 2009;
Vasileva et al. 2009; Kirino et al. 2010), and the interaction
most likely occurred in an sDMA-dependent manner(Kirino et al. 2010). TDRD9 interacts specifically withMIWI2 (Table 2; Shoji et al. 2009; Vagin et al. 2009). TDRD1
may also associate with MIWI2 (Vagin et al. 2009; Wanget al. 2009). Whether the TDRD1–MIWI2 and TDRD9–MIWI2 interactions require sDMAs remains unknown.
sDMA-mediated binding of PIWI proteins with Tud
family members is evolutionarily conserved. Pull-down
assays from Drosophila ovary lysates using synthetic Aub

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Siomi et al.

Table 1. Tud family members in mice
Tud protein

MotifFunction

References

TDRD1/MTR-1

TUDOR 3 4, MYND

Spermatogenesis, piRNA biogenesis

TDRD2/TDRKH
TDRD3
TDRD4/RNF17
TDRD5
TDRD6

TUDOR, KH-I
TUDOR, DUF1767
TUDOR 3 5, BBC
TUDOR
TUDOR 3 7

Implicated in spermatogenesis
Unknown
Implicated in spermatogenesis
Implicated in spermatogenesis
Spermatogenesis, miRNA expression

Chuma et al. 2006;
Reuter et al. 2009
Chen et al. 2009

TDRD7 /TRAPTDRD8/STK31

TUDOR 3 3
TUDOR, polC,
PKc-like
TUDOR, DEXDc,
HELICc, HA2
TUDOR
TUDOR, KH-I
TUDOR, RBB1NT,
ARID, CHROMO
TUDOR, RBB1NT,
ARID, CHROMO
TUDOR 3 2, JmjN,
JmjC, PHD 3 2
TUDOR 3 2, JmjN,
JmjC, PHD 3 2
TUDOR 3 2, JmjN,
JmjC, PHD
LBR-tudor, ICMT

Implicated in spermatogenesis
Implicated in spermatogenesis

Pan et al. 2005
Smith et al. 2004
Hosokawa et al. 2007;
Vasileva et al. 2009
Hosokawa et al. 2007
Chen et al. 2009

Spermatogenesis, piRNA biogenesis

Shoji et al. 2009

Unknown
Mitochondrial metabolism
Implicated in chromatin remodeling

Livigni et al. 2006
Wu et al. 2008

Implicated in chromatin remodeling

Wu et al. 2008

Histone demethylation, self-renewal
in embryonic stem cells
Histone demethylation

Loh et al. 2007

Histone demethylation, self-renewal
in embryonic stem cells
Implicated in morphological
granulocyte maturation
Unknown
Histone methylation, maintenanceof embryonic stem cell state
snRNP assembly
SMN-related protein
Coactivators for signal transducer,
implicated in RNA editing anddegradation, fly Tudor-SN ortholog
Unknown
Unknown

Loh et al. 2007

TDRD9
TDRD12
AKAP1/AKAP121
ARID4A
ARID4B
KDM4A/JMJD2A
KDM4B/JMJD2B
KDM4C/JMJD2C
LBR
MTF2SETDB1/ESETSMN1
SMNDC1
SND1/P100

PHF19
PHF20
PHF20L1ZGPATTUDOR, PHD 3 2
TUDOR 3 2, MBD,
Pre-SET, SET 3 2
TUDOR
TUDOR
TUDOR, SNc 3 5

TUDOR, PHD
TUDOR, MBT, PHD,
DUF3776
TUDOR, MBT, PHD,
DUF3776
TUDOR, zf-CCCH,
G-patchFodor et al. 2006

Cohen et al. 2008

Bilodeau et al. 2009
Gabanella et al. 2005
Talbot et al. 1998
Paukku et al. 2003;
Scadden. 2005

Unknown
Unknown(MYND) Myeloid, Nervy, and DEAF-1 Zinc finger; (KH-I) K homology RNA-binding domain type I; (DUF1767) domain of unknown
function; (SMN) Survival motor neuron protein; (BBC) B-box C-terminal domain; (polC) DNA polymerase III PolC; (PKc-like) protein
kinases catalytic domain; (DEXDc) DEAD-like helicases superfamily; (HELICc) helicase superfamily C-terminal domain; (HA2)helicase-associated domain; (RBB1NT) N terminus to the ARID/BRIGHT domain in DNA-binding proteins of the Retinoblastomabinding protein 1 family; (ARID) ARID/BRIGHT DNA binding domain; (CHROMO) chromatin organization modifier domain; (Jmj)jumonji domain; (PHD) plant homeodomain finger; (LBR-tudor) Lamin-B receptor of TUDOR domain; (ICMT) isoprenylcysteine
carboxyl methyltransferase family; (MBD) methyl-CpG-binding domains; (SET) Su–Enhancer–Trithorax domain; (SNc) staphylococcal
nuclease homologs; (MBT) malignant brain tumor repeat; (DUF3776) domain of unknown function; (zf-CCCH) Zinc finger C-x8-C-x5C-x3-H type; (G-patch) G-patch domain.

peptides, with and without sDMAs, show that a protein
of ;280 kDa associates only with the Aub-sDMA peptide(Kirino et al. 2009; Nishida et al. 2009). This protein was
identified as Tudor by MS, and the association was
confirmed by Western blot analysis using anti-Tud antibodies. Tud members in Drosophila are listed in Table 3.
Of these, CG14303 is the closest homolog of mouse
TDRD1; however, CG14303 does not show an sDMAdependent association with Aub peptides (Nishida et al.

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GENES & DEVELOPMENT

2009). Also, Spn-E, the Drosophila homolog of mouse
TDRD9 (Shoji et al. 2009), shows no detectable association with the peptides (Nishida et al. 2009). Thus,
association of Aub with Tud is rather specific. Similar
assays were performed for AGO3 using peptides that
correspond to two different sDMA-modified regions of
the protein (Nishida et al. 2009). The sDMA peptide
corresponding to the N-terminal end of AGO3 (Fig. 1B)
showed no association with Tud, but the second sDMA

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Tudor in the piRNA pathway

Table 2.

Interaction between PIWI and TDRD proteins in mice

PIWI protein

TDRD protein

References

Adult testis
MILI

TDRD1/MTR-1

MIWI

TDRD2/TDRKH
TDRD6
TDRD1/MTR-1

TDRD7/TRAPTDRD8/STK31

Chen et al. 2009; Kojima et al. 2009;
Reuter et al. 2009; Vagin et al. 2009;
Wang et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Chen et al. 2009; Kojima et al. 2009;
Vagin et al. 2009
Chen et al. 2009; Vagin et al. 2009
Vagin et al. 2009
Chen et al. 2009; Vagin et al. 2009;
Vasileva et al. 2009; Kirino et al. 2010
Chen et al. 2009
Chen et al. 2009

TDRD1/MTR-1
TDRD2/TDRKH
TDRD4/RNF17
TDRD6
TDRD7/TRAPTDRD9
TDRD1/MTR-1
TDRD1/MTR-1
TDRD2/TDRKH
TDRD9Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009
Vagin et al. 2009

TDRD1/MTR-1

TDRD2/TDRKH
TDRD9
TDRD1/MTR-1
TDRD9

Kojima et al. 2009; Reuter et al. 2009;
Vagin et al. 2009; Wang et al. 2009
Vagin et al. 2009; Wang et al. 2009
Vagin et al. 2009
Kojima et al. 2009; Wang et al. 2009;
Vagin et al. 2009
Chen et al. 2009; Vagin et al. 2009
Vagin et al. 2009
Kojima et al. 2009; Wang et al. 2009
Shoji et al. 2009

TDRD6
TDRD6

Vasileva et al. 2009
Vasileva et al. 2009

TDRD2/TDRKH
TDRD4/RNF17
TDRD6Transgenic mouse
Adult testis MIWI (sDMA)

Embryonic testis MILI
Embryonic testis MIWI2

HEK293T/HEK293
MILI (sDMA [Reuter et al. 2009;
Vagin et al. 2009])

MIWI (sDMA [Chen et al. 2009;
Vagin et al. 2009])

MIWI2
Rabbit reticulocyte lysate system
MILI
MIWI

TDRD2/TDRKH
TDRD9
TDRD1/MTR-1

Summary of protein–protein interactions observed between PIWI and TDRD proteins in mice.

peptide, spanning amino acids 58–82 of AGO3, interacted
strongly with Tud. These data indicate clearly that sDMA
modifications alone are not sufficient to promote the
PIWI–Tud association, and that the association occurs ina peptide sequence-specific manner. None of the AGO3
peptides associate with Spn-E (Nishida et al. 2009). AGO3
is considered the Drosophila homolog of MIWI2, in thesense that both proteins interact specifically with secondary piRNAs produced by the amplification loop,
which is operated by Aub and AGO3 in Drosophila andby MILI and MIWI2 in mice (Aravin et al. 2008). Here,a species difference is obvious with regard to the PIWI–
Tud association.
Significance of Tud–PIWI associations
for PIWI–piRNA interactions
Many Tud family members interact with PIWI proteins.
Thus, it can be speculated that this might be the mostprominent pathway for Tud biology. Many groups havelooked into how Tud members affect the association
between PIWI and piRNAs. Immunoprecipitation fromadult mouse testes using an anti-MILI antibody copurified MILI with piRNAs (Kirino et al. 2009; Reuter et al.
2009). In addition, immunoprecipitation using Y12 copurified MILI with piRNAs (Kirino et al. 2009; Reuter et al.
2009). Y12 (antibody) and TDRD1 (protein) recognize thesame sDMA region at the N terminus of MILI; this
implies that a fraction of MILI that is not associated with
TDRD1 is recognized by Y12. This suggests that, even
without association with TDRD1, Mili is able to maintain binding with piRNAs. MILI has additional sDMAs(Fig. 1; Chen et al. 2009; Vagin et al. 2009); however, the
MILI mutant (termed m1), in which R9, R39, R45, andR74 were mutated to lysines (Ks), showed no association
with TDRD1 or TDRD9 (Reuter et al. 2009). Thus,
TDRD1 is not likely to be required for MILI to maintainthe association with piRNAs once the small RNAs have

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Siomi et al.

Table 3. Tud family members in Drosophila
Tud protein

MotifFunction

Closest homologa

References
Schupbach andWieschaus 1986;
Nishida et al. 2009
Pal-Bhadra et al. 2004;
Lim and Kai 2007
Caudy et al. 2003;
Scadden 2005

Tudor

TUDOR 3 11Germline development, piRNAbiogenesis

TDRD6

Spindle E

TUDOR, DEXDc 3 2,
HA2, cas3-core
TUDOR, SNc 3 4

piRNA biogenesis, histon
methylation
RNAi pathway, implicated in
RNA editing and degradation
Unknown
Unknown
Unknown
Unknown
Unknown
Male germline stem cellmaintenancepiRNA biogenesis
Unknown

TDRD9Tudor-SN
CG7082CG8589CG8920
CG13472
CG14303
Female sterile(1) Yb
Krimper
MBD-R2Ovarian tumor

TUDOR, KH-I 3 2
TUDOR
TUDOR 3 3
TUDOR, DUF1767, UBA
TUDOR 3 5
TUDOR, SrmB
TUDOR
TUDOR, THAP,
MBD, PHD
TUDOR, OTU

Polycomblike
Survival motor
neuronYu

TUDOR, PHD 3 2
TUDOR

CG4771
CG9684
CG9925
CG15042
CG15930
CG17454
CG30390CG31755

TUDOR 3 2
TUDOR 3 2, MYNDTUDOR 3 3, MYND
TUDOR
TUDOR
TUDOR
DUF1325
TUDOR, SrmB, DEXDc

TUDOR, KH-I

TDRD8
TDRD2
TDRD5
TDRD7
TDRD3
TDRD1

Szakmary et al. 2009
Lim and Kai 2007

RNA localization nurse cellchromosome dispersion
Chromatin modification
snRNP assembly

Goodrich et al. 2004

Formation of long-term memory,
protein kinase A binding
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown

Lu et al. 2007

Lonie et al. 1994
Miguel-Aliaga et al. 2000

aBased on FlyBase BLAST search.
(DEXDc) DEAD-like helicases superfamily; (HA2) helicase-associated domain; (cas3-core) CRISPR-associated helicase Cas3; (SNc)
staphylococcal nuclease homologs; (KH-I) K homology RNA-binding domain type I; (DUF1767) domain of unknown function; (UBA)ubiquitin-associated domain; (SrmB) superfamily II DNA and RNA helicases; (THAP) thanatos-associated protein domain; (MBD)
methyl-CpG-binding domains; (PHD) plant homeodomain finger; (OTU) ovarian tumor-like cysteine protease; (MYND) myeloid,
Nervy, and DEAF-1 Zinc finger; (DUF1325) SGF29 tudor-like domain. Determined from data in the NCBI HomoloGene and FlyBase
database.

been loaded onto MILI. Whether Y12 copurifies TDRD
proteins with MILI remains undetermined.
Loss of TDRD1 function does not change the ability of
MILI to associate with piRNAs in embryonic testes(Reuter et al. 2009), although the total amounts of piRNAs
are severely decreased in Tdrd1 mutants (Vagin et al.
2009). Deep sequencing analysis revealed that loss of
TDRD1 function leads to overrepresentation of small
RNAs derived from protein-coding transcripts (from bothexons and introns) and of 5S ribosomal RNAs (rRNAs)
associated with MILI (Reuter et al. 2009). Representationof transposon-derived small RNAs is not altered by tdrd1
mutations (Reuter et al. 2009). The molecular mechanismsthat create this discrepancy observed between wild-type
and tdrd1 mutant testes remain unclear. The overallpiRNA profiles in spermatocytes, in contrast, do not seemto be changed by loss of TDRD6 function (Vagin et al.
2009). TDRD6 may also be required for regulation of
miRNAs (Vagin et al. 2009; Vasileva et al. 2009); however,642

GENES & DEVELOPMENT

the effect may be indirect, because changes in developmental timing in tdrd6 mutant mice may result in altered
miRNA profiles, as has been reported by Vagin et al. (2009).
Loss of Tud function in Drosophila ovaries causes Aub
to be associated with a greater abundance of piRNAs
compared with Aub in wild-type ovaries (Nishida et al.
2009). Deep sequencing for piRNAs associated with Aub
in both wild-type and tud ovaries revealed that overrepresentation of any kind of Aub-associated small RNAs
did not occur in tud; however, the population of transposon-derived piRNAs was altered significantly by loss of
Tud function. An obvious change in the strand bias oftransposon-derived piRNAs was not seen in the mutants.
How this alteration is caused by Tud loss remains unclear; however, these results suggest that Tud is required
for the quality control of transposon-derived piRNAs.
Obvious derepression of transposons in tud ovaries has
not been reported. This might be because, although the
population was altered, Aub is still associated with

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Tudor in the piRNA pathway

transposon-derived piRNAs, even in the absence of Tud
function, and thus the Aub–piRNA association state is
not ‘‘perfect’’ but is somewhat ‘‘manageable’’ to silencetransposons, at least in ovaries. However, offspring of tud
mutants are devoid of germ cells. One explanation for thismight be that the phenomenon is simply independent of
the transposon derepression issue.
Depletion of tdrd9 in mice testes causes a massive
increase of LINE1-derived piRNAs, while other piRNAs,
such as the ones derived from IAP and Sine B1 transposons, show a decrease in Tdrd9 mutants (Shoji et al. 2009);
hence, the population of transposon-derived piRNAs ischanged by TDRD9. In this sense, Tud in Drosophila and
TDRD9 in mice are functionally similar to each other,although their similarity at the peptide sequence level is
not particularly high.
Further significant aspects of Tud association
with PIWI proteins
PIWI proteins in Drosophila germlines were originallyreported to be destabilized by loss of sDMA modification(Kirino et al. 2009). However, this interpretation wasrevised later (Nishida et al. 2009; Kirino et al. 2010).
There now seems to be a consensus that the stability of
PIWI proteins is not changed by the loss of sDMA
modification. In tud mutants as well as in prmt5 mutants, the total amounts of Aub and AGO3 were not
reduced, indicating that Tud association with the PIWI
proteins, regardless of the presence of sDMAs, is not
required for their stabilization in vivo. However, thelocalization of PIWI proteins in ovaries was greatly
affected by Tud depletion. In tud mutants, Aub is notdetected at the nuage, although the protein is detected at
the posterior pole of oocytes (Nishida et al. 2009). Kirino
et al. (2010) reported that Aub localization to the posterior pole is markedly reduced in tud mutants. Thus, itremains controversial whether the posterior pole localization of Aub is dependent on its association with Tud.
The protein levels of MILI and its cytoplasmic localization in mouse testes were barely affected by the loss of
TDRD1 expression. However, MIWI2 localization waslargely affected. In wild-type testes, MIWI2 is foundpredominantly in the nucleus and at the nuage. Without
TDRD1, MIWI2 becomes mislocalized, being evenly
distributed in the cytoplasm (Reuter et al. 2009; Shojiet al. 2009; Vagin et al. 2009). The expression level of
MIWI2 was not changed by the loss of TDRD1. Similar
mislocalization of MIWI2 was observed previously in
MILI mutants, where piRNAs were found not to beloaded onto MIWI2 (Aravin et al. 2008). This was because
piRNAs associated with MIWI2 require MILI–Slicer for
their production. This suggested that, without piRNAloading, MIWI2 is not imported into the nucleus. A
possible scenario might then be that, in Tdrd1 mutants,
the amplification loop for producing MIWI2-associatedpiRNAs would be somehow unavailable or not functional, as in mili mutants, and, consequently, this defectfreed MIWI2 from piRNA association, and this absence ofpiRNAs caused MIWI2 to be localized in the cytoplasm.

Indeed, it was found that the levels of piRNAs in theantisense orientation are lowered selectively by TDRD1loss (Vagin et al. 2009). Antisense piRNAs are thought tobe loaded predominantly onto MIWI2 through the amplification loop pathway in mice. Therefore, the decreased
amount of piRNAs associated with MIWI2 might indeed
be the main cause for MIWI2 mislocalization. Loss of
TDRD9 does not cause the mislocalization of MILI,MIWI2, or TDRD1 (Shoji et al. 2009); this is likely to bebecause piRNAs are loaded onto MIWI2, even in thismutant. It seems that the requirements of TDRD1 andTDRD9 in the piRNA production system differ from eachother. In tdrd6 mutants, MIWI was found to be dispersedin the cytoplasm (Vasileva et al. 2009), although the causeof this observation remains unclear.
Earlier studies have revealed that the loss of MILI andMIWI2 causes the expression levels of both LINE1 and
IAP transposons to be much higher compared with thosein wild-type testes (Aravin et al. 2007a; KuramochiMiyagawa et al. 2008). Loss of MILI and MIWI2 impairs
DNA methylation of transposons (Aravin et al. 2007b,
2008; Carmell et al. 2007; Kuramochi-Miyagawa et al.
2008). Interestingly, loss of both TDRD1 and TDRD9
derepresses LINE1 (Reuter et al. 2009; Shoji et al. 2009;
Vagin et al. 2009); however, silencing of IAP remains as inwild type. The DNA methylation state of LINE1, but notof IAP, was also found to be decreased in tdrd1 and tdrd9
mutants (Reuter et al. 2009; Shoji et al. 2009). It is thus
obvious that both TDRD1 and TDRD9 have importantroles, at least in LINE1 silencing in mouse testes.
The current body of evidence shows clearly that sDMA
modifications are made on PIWI proteins by PRMT5.
Also, PIWI proteins associate, via their sDMA modifications, with Tud family members. These mechanisms arehighly evolutionarily conserved and are indispensable forgametogenesis. In fact, genes encoding protein factors
required for this sequential molecular flow are classified
mostly as grandchild-less genes. This simply indicatesthat the system may be germline-specific. Tud family
members have impacts on piRNA production and on
piRNA association with PIWI proteins; however, no
impact on ubiquitously expressed miRNAs or endosiRNAs has so far been reported (except for the possible
involvement of TDRD6 in miRNA regulation). Indeed,none of the ubiquitous AGO proteins that function in
RNA silencing by associating with miRNAs and endosiRNAs have so far been shown to be sDMA-modified, in
contrast to PIWI proteins. Thus, regulation by PRMT5directed sDMA modification and sDMA-specific association with Tud might be a specialized event for the PIWI–
piRNA pathway. Why such an elaborative system exists
specifically for the piRNA pathway remains unknown.
It can be speculated that germlines need to selectively
use PIWI proteins containing sDMA and PIWI without
sDMAs at certain stages during gametogenesis. To clarify
the possibility, it would be interesting to study a dynamic
event for PIWI-sDMA modification in germlines.
How and with what factors are piRNA production andpiRNA association with PIWI proteins regulated? Until
recently, this question was unanswered; however, we

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Siomi et al.

now know that particular members of the Royal Family of
Tudor—TDRD proteins in mice and Tudor in Drosophila—
have roles in piRNA pathways. It is tempting to postulatethat Tud members act as the sovereigns of the whole
piRNA process. Further investigation should help clarify
if this interesting scenario reflects reality, and resolve
other related questions regarding the Royal Family of
Tudor and the piRNA pathways occurring in germlines.
Acknowledgments
We thank Satomi Kuramochi-Miyagawa and Yoichiro Chuma for
useful discussions. H.S. is supported by MEXT (Ministry of
Education, Culture, Sports, Science, and Technology). M.C.S. is
supported by NEDO (New Energy and Industrial Technology
Development Organization) and CREST (Core Research for
Evolutional Science and Technology) from JST (Japanese Scienceand Technology Agency).

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