$FFHSWHG0DQXVFULSW
5HYLHZ)XQFWLRQDODQGVWUXFWXUDOLQVLJKWVLQWRWKHSL51$IDFWRU0DHOVWURP.DRUX6DWR0LNLNR&6LRPL
3,,
'2,
5HIHUHQFH

6  
KWWSG[GRLRUJMIHEVOHW
)(%6

7RDSSHDULQ

FEBS Letters

5HFHLYHG'DWH
$FFHSWHG'DWH

0DUFK
0DUFK

3OHDVHFLWHWKLVDUWLFOHDV6DWR.6LRPL0&)XQFWLRQDODQGVWUXFWXUDOLQVLJKWVLQWRWKHSL51$IDFWRU0DHOVWURPFEBS Letters  GRLKWWSG[GRLRUJMIHEVOHW

7KLVLVD3')ILOHRIDQXQHGLWHGPDQXVFULSWWKDWKDVEHHQDFFHSWHGIRUSXEOLFDWLRQ$VDVHUYLFHWRRXUFXVWRPHUV
ZHDUHSURYLGLQJWKLVHDUO\YHUVLRQRIWKHPDQXVFULSW7KHPDQXVFULSWZLOOXQGHUJRFRS\HGLWLQJW\SHVHWWLQJDQG
UHYLHZRIWKHUHVXOWLQJSURRIEHIRUHLWLVSXEOLVKHGLQLWVILQDOIRUP3OHDVHQRWHWKDWGXULQJWKHSURGXFWLRQSURFHVVHUURUVPD\EHGLVFRYHUHGZKLFKFRXOGDIIHFWWKHFRQWHQWDQGDOOOHJDOGLVFODLPHUVWKDWDSSO\WRWKHMRXUQDOSHUWDLQFunctional and structural insights into the piRNA factor Maelstrom
Kaoru Sato and Mikiko C. Siomi*
Department of Biological Sciences, Graduate School of Science, The University ofTokyo, Tokyo 113-0032, Japan*Correspondence: siomim@bs.s.u-tokyo.ac.jp

Abstract
PIWI-interacting RNA (piRNA) is a germline-specific class of small non-coding RNAs
that repress transposons in the gonads. Mael, which comprises a high mobility group box
and a MAEL domain, is one of the key players in piRNA-mediated transposon silencing.
However, the mechanism whereby Mael is involved in this pathway remains unknown.
Recent biochemical and structural studies, along with bioinformatic analyses ofMael-associating RNAs in vivo, have shed light on the functional aspects of Mael in thepiRNA pathway. We summarize the current understanding of Mael functions in thepiRNA pathway, particularly in Drosophila and in mice.

Key words: piRNA; PIWI; Maelstrom; MTOC; transposon silencing

1Introduction
Gene silencing mechanisms triggered by small, 20-33 nucleotide RNA are collectively
called RNA silencing, which is involved in numerous biological events in a variety oforganisms [1-6]. In RNA silencing, small RNAs associate with members of the Argonautefamily of proteins to form RNA-induced silencing complexes (RISCs), which are guided
to target transcripts through RNA-RNA base pairing [1-6]. Potent RNA silencing smallRNAsinclude smallinterfering

RNAs(siRNAs),

microRNAs(miRNAs) andPIWI-interacting RNAs (piRNAs). Argonaute proteins are divided into AGO and PIWI
subfamilies, depending on amino acid sequence similarity and tissue specificity [1-3].
Ubiquitous AGO proteins bind siRNAs and miRNAs to assemble siRISCs and miRISCs,
respectively, whereas germline-specific PIWI proteins bind piRNAs to form piRISCs[6-8].
Like its protein partner PIWI, piRNA is germline specific. Deep sequencing of piRNAs in
the germlines of various animals such as Drosophila, fish, frogs, mice, and marmosets
showed that the majority of piRNAs have sequences that are complementary totransposons [9-15]. Transposons move around within the genome and therefore havethe potential to cause genomic damage, which often induces harmful disorders including
aberrant

chromosomal

rearrangements

anddevelopmental

defects[6,

16].

Rerepression of transposons is therefore essential for living organisms, particularly in
the germline; not surprisingly, the piRNA pathway is therefore highly conserved inanimals with sexual reproduction systems.
piRNA precursors are transcribed from piRNA clusters, which are intergenic elementsrich in transposon fragments and/or other types of repetitive sequences [9, 17]. piRNA
precursor processing is extremely intricate and the underlying mechanism is notcompletely understood. After processing, mature piRNAs are specifically loaded onto
PIWI proteins (although AGO proteins are also present in the germlines) [6, 18-25].
2

piRNA processing and piRISC formation are thought to occur at cytoplasmic loci termed
nuage (French for “cloud”), chromatoid bodies, P-bodies or piP-bodies in germ cells, andYb bodies in Drosophila ovarian somatic cells [6, 26-42]. This specific localization likely
contributes to strict sorting of piRNAs onto PIWI proteins.
Based on high-throughput sequencing and bioinformatic analyses of piRNAs in various
animals, two piRNA biogenesis pathways have been proposed, the primary processing
pathway and the ping-pong amplification loop [6, 9, 10]. Germ cells in Drosophila ovaries
express three PIWI proteins, Piwi, Aubergine (Aub), and AGO3; primary piRNAs
produced through the primary processing pathway are loaded onto Piwi and Aub, but not
onto AGO3 [6, 9, 10, 19, 23, 43]. Piwi-piRISC localizes to the nucleus upon its
cytoplasmic assembly and triggers transcriptional silencing of transposons [6, 11].
Meanwhile, cytoplasmic Aub-piRISC initiates the ping-pong cycle with AGO3 byreciprocally cleaving target RNAs using an endonuclease (slicer) activity to produce
secondary piRNAs in the germlines [6, 11, 43]. The ping-pong cycle, which consumes
transposon transcripts to provide a secondary piRNA pool, is a coupled event consistingof post-transcriptional transposon silencing and piRNA biogenesis. Somatic follicle cells
express Piwi, but not Aub and AGO3, and therefore primary piRNAs are solely loaded
onto Piwi [6, 11, 12, 43]. As in germ cells, Piwi-piRISC in somatic cells silencetransposons transcriptionally after assembling larger complexes with multiple co-factors
in the nucleus [12, 44, 45].
Mice express three PIWI proteins, MIWI, MILI, and MIWI2 in the testis in a
spatiotemporal-specific manner [46]. In gonocytes, MILI and MIWI2, but not MILI2, areexpressed, where MILI associates with primary piRNAs derived from prepachytene
piRNA clusters, and cleaves target RNAs in the cytoplasm that produces secondary
piRNAs for MIWI2. MIWI2 then localizes to the nucleus where it implements
transcriptional transposon silencing through de novo DNA methylation. De novo DNA
3methylation does not occur in Drosophila because the species lacks the enzyme
responsible for the modification. At the pachytene stage, where MIWI2 is no longer
expressed, MIWI and MIWI2 associate with primary piRNAs arising from pachytene
piRNA clusters, and implement post-transcriptional silencing by cleaving target RNAs in
the cytoplasm.
To date, a number of factors necessary for both the initiation (i.e., piRNA biogenesis) andeffector (i.e., piRNA-mediated silencing) phases of the piRNA pathway, have been
identified, many of which are evolutionarily conserved [42, 47-51]. Of those, Maelconsists of a high mobility group (HMG) box and a MAEL domain at the N-terminal andcentral regions, respectively [44, 52, 53]. Mael was originally identified as aP-element-induced female sterile mutant that affects axis specification in Drosophila
oocytes [52]. However, genetic studies have indicated that loss of Mael causes defects
in piRNA accumulation and transposon silencing in Drosophila ovaries [29]. The link
between oocyte axis specification and piRNA-mediated transposon silencing (both ofwhich are dependent of Mael), as well as the mechanistic involvement of Mael in thepiRNA pathway remains largely unclear. However, thanks to the insights into Maelfunction gained from recent studies, the model of piRNA pathway in animals has been
improved.

Mael is a MTOC component in Drosophila ovaries
The localization of RNA transcripts (including Bicoid (bcd), Oskar (osk), and a ligand forthe Drosophila Egf receptor Gurken (grk)) to the posterior end of developing oocytes
plays a pivotal role in embryogenesis. The process depends on polarization of the
microtubule cytoskeleton and correlates with the formation of a microtubule-organizing
center (MTOC), a structure from which most microtubules grow [54-62]. Meanwhile,
mutations in Mael disturb the localization of mRNAs in developing oocytes because of4

improper placement and/or anchoring of the MTOC, because the MTOC fails to movefrom the anterior to the posterior end of the oocytes in Mael mutants [63]. Indeed,mutations in Mael prevent clustering of centrioles and posterior localization of the MTOC
in stage 2-6 oocytes, indicating the involvement of Mael, directly or indirectly, in MTOC
formation [63]. Centrosomes act as MTOCs that nucleate microtubules with uniform
polarity and consist of a pair of centrioles that recruit a large number of proteins,collectively referred to as pericentriolar materials. Of these, six proteins, Centrosomin(Cnn), Drosophila transforming acidic coiled-coil protein (D-TACC), Mini spindles (Msps),
Short spindle 2 (Ssp2), dNAT1, and No circadian temperature entrainment (Nocte), wereidentified as major Mael-interacting proteins [64-74]. A clear link therefore exists
between Mael and the MTOC.
Mael also co-localizes with alpha- and gamma-tubulin in both dividing cysts in the
germarium and in dividing follicle cells in ovarioles, indicating that Mael preferentiallylocalizes to microtubule spindles [64]. In Mael mutant ovaries, abnormal egg chamberswith reduced cell numbers are generated because cytoplasmic mitotic divisions arearrested [64]. Moreover, gamma-tubulin and Cnn are mis-localized or abnormally
accumulated in Mael mutant egg chambers, which suggests functional involvement ofMael in proper localization and expression of MTOC proteins [64]. Thus, Mael nucleates
other MTOC components to form a functional MTOC during Drosophila oogenesis.
Mutations in genes belonging to the Drosophila piRNA pathway may lead to germline
DNA damage and disruption of embryonic axis specification through activation of
ATM/Chk2, which functions in DNA damage response signalling [75-78]. During
oogenesis, DNA damages (such as double-strand DNA breaks) caused by de novotransposon insertions, are met with activation of Mei-41 and Mnk that encode ATR and
Chk2 kinases, respectively; Chk2 then blocks axis specification by disrupting
microtubule organization and phosphorylating Vasa [78-80]. Vasa is a DEAD-box RNA
5helicase required for axis specification that has been implicated in Grk mRNA translation[78]. Embryonic axis specification defects (which have been linked to several piRNA
pathway mutations) are genetically suppressed by a loss-of-function mutation in Mnk,
which is required for DNA damage signaling [78-80]. Loss of Mael function disrupts theposterior localization of Osk and the dorsal anterior localization of Grk in the oocyte [64].
However, unlike other piRNA pathway mutations, the localization of Osk and Grk is notrestored in Mnk;Mael double mutants [64]. Therefore, defects in microtubuleorganization observed in the Mael oocyte may have distinct origins comparable with
DNA damage caused by defects in the piRNA pathway.

The role of Mael in transcriptional silencing in Drosophila ovarian somatic cells
Piwi functions in transcriptional transposon silencing in both germ and ovarian somatic
cells in Drosophila [9-12, 25, 44]. Piwi-mediated transposon silencing depends on Piwi’s
nuclear localization and piRNA loading, but not on its slicer activity [32, 44, 81, 82].
Transposons targeted by Piwi in ovarian somatic cells are classified into groups I, II, andIII [44]. The expression of group I transposons are greatly affected by loss of Piwi, whilethat of group II transposons are only mildly affected, and group III transposons exhibit
little change upon loss of Piwi [44]. Piwi-bound antisense piRNA levels are typically highfor transposons in groups I and II, but little for group III transposons [44]. Thus,
Piwi-mediated silencing depends on its piRNA pool.
In Piwi-depleted cells, RNA Polymerase II (Pol II) occupancy increases significantly at
the genomic loci of group I and II transposons, but not in group III transposons. Further,group I transposons exhibit low levels of histone H3 lysine 9 trimethylation (H3K9me3), a
major repressive histone mark [44]. These results provide sufficient proof that
Piwi-mediated silencing occurs at the transcriptional, but not the post-transcriptional

6

level [44]. Loss of Piwi also leads to up-regulation of neighboring genes, which suggeststhat Piwi-piRISCs trigger local H3K9me3 spreading [44].
In Mael mutant ovaries, piRNA levels are reduced, and as a consequence, transposons
are derepressed [29]. By contrast, in cultured Drosophila ovarian somatic cells (OSCs)[12, 83], Mael deletion by RNAi does not affect piRNA biogenesis, but results inderepression of transposons [7, 32, 44]. Thus, Mael acts during the effector phase of thesomatic piRNA pathway [84]. Indeed, knocking down Mael results in derepression ofgroup I and II transposons similar to Piwi knockdowns [44]. Likewise, Piwi and Mael
double knockdowns exhibit transposon derepression similar to Piwi single knockdowns,
further supporting the idea that both proteins act in the same pathway [44]. Although Piwi
and Mael depletion increases transcription and Pol II occupancy of transposons in a very
similar manner, changes in H3K9me3 patterns differ significantly in cells where Piwi andMael were depleted. H3K9me3 at transposon loci is slightly reduced in Mael knockdown
cells. Thus, Mael apparently acts downstream of Piwi and the establishment of
H3K9me3 [44].

The MAEL domain in Mael exhibits endoribonuclease activity
The MAEL domain is predicted to adopt an RNase H-like fold [53]. Recently, the X-ray
crystal structure of the MAEL domain of Drosophila and Bombyx Mael has been solved(Figure 1). The Drosophila MAEL domain consists of a twisted five-stranded mixed
β-sheet surrounded by 13 helices, with a zinc ion coordinated by the ECHC motif [85].
The structure of Bombyx MAEL domain shows a high similarity to that of Drosophila Mael[86]. The ECHC motif is highly conserved in MAEL domains in animals [85]. Mutations in
the ECHC motif destabilize the Mael protein, which demonstrates that zinc ion
coordination by the ECHC amino acid tetrad is indispensable for proper functioning ofMael [85]. Interestingly, the MAEL domain of Mael lacks canonical catalytic residues
7

DEDDh conserved among RNase H-like superfamily members [85]. However,
unexpectedly, it exhibits a single-stranded (ss) endoribonuclease (RNase) activity invitro [85]. The cleavage pattern of ssRNA substrates by the MAEL domain is similar tothat of RNase T1, a well-studied endonuclease that specifically cleaves ssRNA at the 3’
side of guanine residues [87]. However, unlike RNase T1, the ssRNase activity of the
MAEL domain does not require divalent metal ions, but is inhibited by high salt [85]. The
RNA cleavage mechanism of the MAEL domain is distinct from that of RNase T1.
Notably, the ssRNase activity is also observed in the MAEL domain of Bombyx andmouse Mael proteins in vitro, at least under condition of a low salt [85].
Mutations in several conserved residues among orthologs of the MAEL domain severely
reduced, but did not abolish its ssRNase activity, implying that those residues might beinvolved in ssRNA binding, but not catalysis. Neither the catalytic center nor themechanism of catalysis has been uncovered to date. One could argue that the MAEL
domain would have evolved from a DEDDh exonuclease by switching the catalytic
residues from DEDDh to ECHC (Figure 1) [53, 85]. The ECHC tetrad is highly conserved
among Mael in various species, which suggest that this tetrad might be the active center.
However, because the tetrad is responsible for maintaining both protein structure andnuclease activity, this would be too difficult to prove experimentally.
The MAEL domain of Drosophila Mael is able to restore silencing defects caused bydepletion of endogenous Mael in OSC cells, which means that the HMG box isdispensable for nuclear silencing of transposons in the cells (Figure 2) [7, 44, 85].
However, MAEL domain mutants that reduce the ssRNase activity of Mael have also
been shown to rescue the silencing defects in OSCs [85]. The relevance of Mael’s
ssRNase activity in piRNA-mediated silencing therefore remains unclear. Whether the
ssRNase activity of the MAEL domain is required for piRNA biogenesis in Drosophila
ovarian somatic cells is not yet known.
8

Mael in adult mouse testis binds pachytene piRNA precursors
Based on microarray-based expression profiling in mice, Mael is necessary for earlyspermatogenesis [88]. Mael is highly expressed in the testis, but its cellular localization
changes through spermatogenesis; in spermatocytes it is cytoplasmic while it localizes in
the nucleus in spermatids [87]. MAEL is concentrated at the sex body in the nucleus andchromatoid body/nuage in the cytoplasm [88-90]. The sex body is a specialized nuclear
area where both transcription and homologous recombination are restricted duringmeiosis, whereas the chromatoid body/nuage is a unique cloud-like dense structure
found in male germ cells, where various piRNA factors accumulate [31, 33, 34, 38, 39, 41,46]. Unlike Mael in Drosophila, Mael in mice interacts with Miwi and Mili, two of threemurine PIWI proteins, in the adult mouse testis [88]. MIWI and Mili (also known as
PIWIL1 and PIWIL2), but not Miwi2 (PIWIL4), are expressed in the adult mouse testis [6,31, 34, 46]. The Mael knockout mouse testis appears normal anatomically, with the
exception of smaller testes [89]. In the absence of Mael, post-meiotic germ cells (roundand elongated spermatids) were completely absent, demonstrating the necessity of Maelin spermatogenesis [89]. In addition, long interspersed nucleotide element (LINE)-1 andintracisternal A particle (IAP) element, non-LTR and LTR retrotransposons, respectively,
are derepressed, and CpG DNA methylation levels associated with LINE-1 genomic
regions are significantly reduced in loss of Mael testes [89]. Overall, Mael knockout micephenocopy MILI and MIWI2 knockout mice: a close functional relationship of MAEL withthese two PIWI proteins was suggested. Recently, analysis of the Mael-associatedproteome revealed that Mael is strongly associated with MIWI and a Tudor
domain-containing protein TDRD6 in adult mouse testes (Figure 2) [91, 92]. Three other
Tudor proteins TDRD1, TDRD4/RNF17, and TDRD9 also appeared in the
Mael-associated proteome. MILI was detected in the adult mouse testis, although, at a9

lower percent coverage compared with MIWI. An earlier report showed that a DEAD-box
RNA helicase Mouse Vasa homolog (MVH) associates with MIWI and TDTD6 in adulttestes [93, 94]. However, Mael complexes rich in MIWI and TDTD6 appeared to beabsent of MVH [91]. Mael interaction with MIWI and Tdrd6 is likely RNA independent.
Examination of RNA components in the Mael complex revealed pachytene piRNA
precursors (Figure 2) [91]. In addition, the Mael complex is also enriched with transcripts
of known and hypothetical protein-coding genes, pseudogenes, as well as non-coding
RNAs. Because the Mael complex is devoid of mature piRNAs, Mael may play animportant role in the pachytene piRNA processing step. Indeed, loss of Mael in mousetestes reduces the level of pachytene piRNAs in the testis. A recent study showed that
the MAEL domain of mouse and Drosophila Mael exhibits an ssRNase activity in vitro[85]. Further studies are needed to determine whether Mael is responsible for pachytene
piRNA processing in the adult mouse testis. The HMG box in mouse Mael preferentiallybinds structured RNAs [95]. Given that mouse Mael is found in both the nucleus and thecytoplasm [88], and that Mael is a shuttling protein [28], Mael may bind pachytene piRNA
primary precursors in the nucleus through its HMG box and export them to the cytoplasm,
where the precursors accumulate prior to being processed into mature piRNAs (Figure2) [92].

Perspective
Mael is a conserved piRNA factor in animals, consisting of two domains, a HMG box anda MAEL domain. The MAEL domain of Mael has ssRNase activity. In Drosophila ovariansomatic cells, Mael acts as an effector of the Piwi-mediated transcriptional silencing oftransposons; only the MAEL domain, and not the HMG box of Mael is required for thissilencing. In addition, its ssRNase activity is not required for silencing transposons insomatic cells. These observations imply that the MAEL domain acts as a scaffold for10

Piwi-nuclear complexes to effectively silence transposons in Drosophila soma. In
contrast, Mael is involved in the piRNA production in germ cells. Moreover, the HMG box
of Mael is dispensable for silencing in somatic cells but is required for the piRNA
production in germ cells. The cell-specific dual functions of Mael in the piRNA pathway is
a unique aspect of Mael.
Functional analyses of mouse Mael may provide good clues to understanding themolecular functions of Mael in piRNA production in Drosophila germ cells. The mouse
Mael complex, which contains MIWI and TDRD6, is enriched with pachytene piRNA
primary precursors, where precursors are undergoing fragmentation/processing into
mature piRNAs. Because the mouse HMG box has binding activity to structured RNAs, it
seems likely that pachytene piRNA primary precursors may be anchored by the HMG
box and then fragmented by the MAEL domain. In Drosophila germ cells, Mael isinvolved in the production of piRNAs, and the HMG box of Mael is required for
germline-specific transposon silencing. These results imply that Drosophila Mael also
performs similar functions to mouse Mael. Further biochemical studies are needed toshed light on the molecular roles of Mael in piRNA pathways.

Acknowledgements
We thank O. Nureki, H. Nishimasu, and N. Matsumoto at the University of Tokyo forsharing the Drosophila MAEL structure. We also thank H. Siomi at Keio University
School of Medicine for comments on the manuscript. This work is supported by the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) and
Grants-in-Aid for Scientific Research to K.S. and M.C.S.

11

References
1. Ghildiyal, M. & Zamore, P.D. (2009). Small silencing RNAs: an expanding universe.
Nat. Rev. Genet. 10, 94-108.
2. Kim, N.V., Han, J. & Siomi M.C. (2009).Biogenesis of small RNAs in animals. Nat.
Rev. Mol. Cell Biol. 10, 126-139.
3. Malone, C.D. & Hannon, G.J. (2009).Small RNAs as guardians of the genome. Cell
136, 656-668.
4. Siomi, H. & Siomi, M.C. (2009). On the road to reading the RNA-interference code.
Nature 457, 396-404.
5. Thomson, T. & Lin, H. (2009). The biogenesis and function of PIWI proteins andpiRNAs: progress and prospect. Annu. Rev. Cell Dev. Biol. 25, 355-376.
6. Siomi, M.C., Sato, K., Pezic, D., Aravin, A.A. (2011). PIWI-interacting small RNAs:
The vanguard of genome defence. Nat. Rev. Mol. Cell. Biol. 12, 246-258.
7. Ohtani, H., Iwasaki, Y.W., Shibuya, A., Siomi, H., Siomi, M.C., Saito, K. (2013).
DmGTSF1 is necessary for Piwi-piRISC-mediated transcriptional transposon
silencing in the Drosophila ovary. Genes Dev. 27, 1656-1661.
8. Murota, Y., Ishizu, H., Nakagawa, S., Iwasaki, Y.W., Shibata, S., Kamatani, M.K.,
Saito, K., Okano, H., Siomi, H., Siomi, M.C. (2014). Yb integrates piRNAintermediates and processing factors into perinuclear bodies to enhance piRISC
assembly. Cell Rep. 8, 103-113.
9. Brennecke, J. Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R.,
Hannon, G.J. (2007). Discrete small RNA-generating loci as master regulators oftransposon activity in Drosophila. Cell 128, 1089–1103.
10. Gunawardane, L.S. Saito, K., Nishida, K.M., Miyoshi, K., Kawamura, Y., Nagami, T.,
Siomi, H., Siomi, M.C. (2007). A slicer-mediated mechanism for repeat-associatedsiRNA 5’ end formation in Drosophila. Science 315, 1587̽1590.
11. Malone, C.D., Brennecke, J., Dus, M., Stark, A., McCombie, W.R., Sachidanandam,R., Hannon, G.J. (2009). Specialized piRNA pathways act in germline and somatic
tissues of the Drosophila ovary. Cell.137, 522-535.
12. Saito, K., Inagaki, S., Mituyama, T., Kawamura, Y., Ono, Y., Sakota, E., Kotani, H.,
Asai, K., Siomi, H., Siomi, M.C. (2009). A regulatory circuit for piwi by the large Maf
gene traffic jam in Drosophila. Nature 461, 1296–1299.
13. Houwing, S., Kamminga, L.M., Berezikov, E., Cronembold, D., Girard, A., van den
Elst, H., Filippov, D.V., Blaser, H., Raz, E., Moens, C.B., Plasterk, R.H., Hannon, G.J.,
Draper, B.W., Ketting, R.F. (2007). A role for Piwi and piRNAs in germ cellmaintenance and transposon silencing in Zebrafish. Cell 129, 69–82.
14. Lau, N.C., Ohsumi, T., Borowsky, M., Kingston, R.E., Blower, M.D. (2009).
Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi.
EMBO J. 28, 2945-2958.
15. Hirano, T., Iwasaki, Y.W., Lin, Z.Y., Imamura, M., Seki, N.M., Sasaki, E., Saito, K.,
Okano, H., Siomi, M.C., Siomi, H. (2014). Small RNA profiling and characterization ofpiRNA clusters in the adult testes of the common marmoset, a model primate. RNA.
20, 1223-1237.
16. Kazazian, H.H.Jr. (2004). Mobile elements: drivers of genome evolution. Science
303, 1626-1632.
17. Yamanaka, S., Siomi, M.C., Siomi, H. (2014). piRNA clusters and open chromatin
structure. Mobile DNA. 5, 22.
18. Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V. & Zamore, P.D. (2006). A
distinct small RNA pathway silences selfish genetic elements in the germline.
Science 313, 320–324.
1219. Saito, K., Nishida, K.M., Mori, T., Kawamura, Y., Miyoshi, K., Nagami, T., Siomi, H.,
Siomi, M.C. (2006). Specific association of Piwi with rasiRNAs derived fromretrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev.
20, 2214–2222.
20. Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N.,
Morris, P., Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T., Chien, M., Russo,
J.J., Ju, J., Sheridan, R., Sander, C., Zavolan, M., Tuschl, T. (2006). A novel class of
small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207.
21. Girard, A., Sachidanandam, R., Hannon, G. J. & Carmell, M.A. (2006). A
germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442,
199–202.
22. Grivna, S. T., Beyret, E., Wang, Z., Lin, H. (2006). A novel class of small RNAs in themouse spermatogenic cells. Genes Dev. 20, 1709–1714.
23. Nishida, K.M., Saito, K., Mori, T., Kawamura, Y., Nagami-Okada, T., Inagaki, S.,
Siomi, H., Siomi, M.C. (2007). Gene silencing mechanisms mediated by Aubergine
piRNA complexes in Drosophila male gonad. RNA 13, 1911–1922.
24. Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V., Zamore, P.D. (2006). A Distinct
small RNA pathway silences selfish genetic elements in the germline. Science 313,
320–324.
25. Saito, K., Nishida, K.M., Mori, T., Kawamura, Y., Miyoshi, K., Nagami, T., Siomi, H.,
Siomi, M.C. (2006). Specific association of Piwi with rasiRNAs derived fromretrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev.
20, 2214–2222.
26. Eddy, E.M. (1975). Germ plasm and the differentiation of the germ line. Int. Rev.
Cytol. 43, 229 -281.
27. Saffman, E.E. & Lasko, P. (1999). Germline development in vertebrates andinvertebrates. Cell Mol. Life Sci. 55, 1141-1163.
28. Findley, S.D., Tamanaha, M., Clegg, N.J., Ruohola-Baker, H. (2003). Maelstrom, aDrosophila spindleclass gene, encodes a protein that colocalizes with Vasa and
RDE1/AGO1 homolog, Aubergine, in nuage. Development 130, 859–871.
29. Lim, A.K & Kai, T. (2007). Unique germ-line organelle, nuage, functions to repress
selfish genetic elements in Drosophila melanogaster. Proc. Natl Acad. Sci. USA 104,
6714–6719.
30. Nagao, A., Sato, K., Nishida, K.M., Siomi, H., Siomi, M.C. (2011). Gender-Specific
Hierarchy in Nuage Localization of PIWI-Interacting RNA Factors in Drosophila.
Front. Genet. 2, 55.
31. Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa, A., Tadokoro, Y.,
Fujita, Y., Sato, M., Matsuda, Y., Nakano, T. (2001). Two mouse piwi-related genes:
miwi and mili. Mech. Dev. 108, 121–133.
32. Saito, K., Ishizu, H., Komai, M., Kotani, H., Kawamura, Y., Nishida, K.M., Siomi, H.,
Siomi, M.C. (2010). Roles for the Yb body components Armitage and Yb in primarypiRNA biogenesis in Drosophila. Genes Dev. 24, 2493-2498.
33. Vasileva, A., Tiedau, D., Firooznia, A., Müller-Reichert, T., Jessberger, R. (2009).
Tdrd6 is required for spermiogenesis, chromatoid body architecture, and regulationof miRNA expression. Curr. Biol. 19, 630–639.
34. Aravin, A.A., Sachidanandam, R., Bourc'his, D., Schaefer, C., Pezic, D., Toth, K.F.,
Bestor, T., Hannon, G.J. (2008). A piRNA pathway primed by individual transposons
is linked to de novo DNA methylation in mice. Mol. Cell 31, 785–799.
35. Qi, H., Watanabe, T., Ku, H.Y., Liu, N., Zhong, M., Lin, H. (2011). The Yb body, amajor site for Piwi-associated RNA biogenesis and a gateway for Piwi expression
and transport to the nucleus in somatic cells. J. Biol. Chem. 286, 3789-3797.
1336. Ma, L., Buchold, G.M., Greenbaum, M.P., Roy, A., Burns, K.H., Zhu, H., Han, D.Y.,
Harris, R.A., Coarfa, C., Gunaratne, P.H., Yan, W., Matzuk, M.M. (2009). GASZ isessential for male meiosis and suppression of retrotransposon expression inthe malegermline. PLoS Genet. 5, e1000635.
37. Kotaja, N., Lin, H., Parvinen, M., Sassone-Corsi, P. (2006). Interplay of
PIWI/Argonaute protein MIWI and kinesinKIF17b in chromatoid bodies of male germ
cells.J. Cell Sci. 119, 2819–2825.
38. Kotaja, N., Bhattacharyya, S.N., Jaskiewicz, L., Kimmins, S., Parvinen, M., Filipowicz,
W., Sassone-Corsi, P. (2006). The chromatoid body of male germ cells: similarity
with processing bodies and presence of Dicer and microRNA pathway components.
Proc. Natl Acad. Sci. USA 103, 2647–2652.
39. Onohara, Y., Fujiwara, T., Yasukochi, T., Himeno, M., Yokota, S. (2010). Localizationof mouse vasa homolog protein in chromatoid body and related nuage structures ofmammalian spermatogenic cells during spermatogenesis. Histochem. Cell Biol. 133,
627–639.
40. al-Mukhtar, K. A. & Webb, A.C. (1971). An ultrastructuralstudy of primordial germ
cells, oogonia and early oocytes in Xenopus laevis. J. Embryol. Exp. Morphol.26,
195–217.
41. Yadav, R.P., & Kotaja, N. (2014). Small RNAs in spermatogenesis. Mol Cell
Endocrinol. 382, 498-508.
42. Ishizu, H., Siomi, H., Siomi, M.C. (2012).Biology of PIWI-interacting RNAs: new
insights into biogenesis and function inside and outside of germlines. Genes Dev. 26,
2361-2373.
43. Li, C., Vagin, V.V., Lee, S., Xu, J., Ma, S., Xi, H., Seitz, H., Horwich, M.D., Syrzycka,
M., Honda, B.M., Kittler, E.L., Zapp, M.L., Klattenhoff, C., Schulz, N., Theurkauf,
W.E., Weng, Z., Zamore, P.D. (2003). Collapse of germline piRNAs in the absence of
Argonaute3 reveals somatic piRNAs in flies. Development 30, 859-871.
44. Sienski, G., Donertas, D., Brennecke, J. (2012). Transcriptional silencing oftransposons by Piwi and maelstrom and its impact on chromatin state and gene
expression. Cell. 151, 964–980.
45. Watanabe, T. & Lin, H. (2014). Posttranscriptional regulation of gene expression by
Piwi proteins and piRNAs. Mol. Cell. 56, 18-27.
46. Siomi, M.C. & Kuramochi-Miyagawa, S. (2009). RNA silencing in germlines –exquisite collaboration of Argonaute proteins with small RNAs for germline survival.
Curr. Opin. Cell Biol. 3, 426-434
47. Saito, K. & Siomi, M.C. (2010). Small RNA-mediated quiescence of transposable
elements in animals. Dev. Cell 19, 687–697.
48. Siomi, M.C., Mannen, T., Siomi, H. (2010). How does the royal family of Tudor rule
the PIWI-interacting RNA pathway? Genes Dev. 24, 636-646.
49. Handler, D., Meixner, K., Pizka, M., Lauss, K., Schmied, C., Gruber, F.S., Brennecke,
J. (2013). The genetic makeup of the Drosophila piRNA pathway. Mol Cell
50:762-777.
50. Czech, B., Preall, J.B., McGinn, J., Hannon, G.J. (2013). A transcriptome-wide RNAi
screen in the Drosophila ovary reveals factors of the germline piRNA pathway. Mol
Cell. 50, 749-761.
51. Muerdter, F., Guzzardo, P.M., Gillis, J., Luo, Y., Yu, Y., Chen, C., Fekete, R., Hannon,
G.J. (2013). A genome-wide RNAi screen draws a genetic framework for transposon
control and primary piRNA biogenesis in Drosophila. Mol Cell. 50, 736-748.
52. Clegg, N.J., Frost, D.M., Larkin, M.K., Subrahmanyan, L., Bryant, Z., Ruohola-Baker,
H. (1997). maelstrom is required for an early step in the establishment of Drosophila
oocyte polarity: Posterior localization of grk mRNA. Development 124, 4661–4671.
1453. Zhang, D., Xiong, H., Shan, J., Xia, X., Trudeau, V.L. (2008). Functional insight into
Maelstrom in the germline piRNA pathway: A unique domain homologous to the
DnaQ-H 3’̽5’ exonuclease, its lineage-specific expansion/loss and evolutionarily
active site switch. Biol. Direct 3, 48.
54. Ephrussi, A., Dickinson, L.K., Lehmann, R. (1991). oskar organizes the germ plasm
and directs localization of the posterior determinant nanos. Cell 66, 37–50.
55. Chasan, R., Anderson, K. (1993). Maternal control of dorso–ventral polarity andpattern in the embryo. In The development of Drosophila melanogaster (ed. Bate M.
and Martínez-Arias A.), pp. 387–424. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
56. Neuman-Silberberg, F.S. & Schupbach, T. (1993). The Drosophila dorsoventral
patterning gene gurken produces a dorsally localized RNA and encodes a TGF α-likeprotein. Cell 75, 165–174.
57. Pokrywka, N., Stephenson, E. (1995). Microtubules are a general component ofmRNA localization systems in Drosophila oocytes. Dev. Biol. 167, 363–370.
58. van Eeden, F., St Johnston, D. (1999). The polarisation of the anterior–posterior anddorsal–ventral axes during Drosophila oogenesis. Curr. Opin. Genet. Dev. 9,396–404.
59. Riechmann, V., Ephrussi, A. (2001). Axis formation during Drosophila oogenesis.
Curr. Opin. Genet. Dev. 11, 374–383.
60. Theurkauf, W., Alberts, M., Jan, Y., Jongens, T. (1993). A central role formicrotubules in the differentiation of Drosophila oocytes. Development 118,
1169–1180.
61. Januschke, J., Gervais, L., Gillet, L., Keryer, G., Bornens, M., Guichet, A. (2006). The
centrosome–nucleus complex and microtubule organization in the Drosophila oocyte.
Development 133, 129–139.
62. Steinhauer, J. & Kalderon, D. (2006). Microtubule polarity and axis formation in the
Drosophila oocyte. Dev. Dyn. 235, 1455–1468.
63. Clegg, N.J., Findley, S.D., Mahowald, A.P., Ruohola-Baker, H. (2001). Maelstrom isrequired to position the MTOC in stage 2–6 Drosophila oocytes. Dev Genes Evol 211,44–48.
64. Sato, K., Nishida, K.M., Shibuya, A., Siomi, M.C., Siomi, H. (2011). Maelstrom
coordinates microtubule organization during Drosophila oogenesis throughinteraction with components of the MTOC. Genes Dev. 25, 2361-2373.
65. Kwon, M. & Scholey, J.M. (2004). Spindle mechanics and dynamics during mitosis in
Drosophila. Trends Cell Biol 14, 194–205.
66. Megraw, T.L., Kao, L.R., Kaufman, T.C. (2001). Zygotic development without
functional mitotic centrosomes. Curr. Biol. 11, 116–120.
67. Terada, Y., Uetake, Y., Kuriyama, R. (2003). Interaction of aurora-A and centrosomin
at the microtubule-nucleating site in Drosophila and mammalian cells. J Cell Biol 162,
757–764.
68. Cullen, C.F. & Ohkura, H. (2001). Msps protein is localized to acentrosomal poles toensure bipolarity of Drosophila meiotic spindles. Nat. Cell. Biol. 3, 637–642.
69. Cullen, C.F., Deák, P., Glover, D.M., Ohkura, H. (1999). Mini spindles: A geneencoding a conserved microtubule-associated protein required for the integrity of themitotic spindle in Drosophila. J. Cell. Biol. 146, 1005–1018.
70. Gergely, F., Kidd, D., Jeffers, K., Wakefield, J.G., Raff, J.W. (2000). D-TACC: A
novel centrosomal protein required for normal spindle function in the early Drosophila
embryo. EMBO J. 19, 241–252.

15

71. Lee, M.J., Gergely, F., Jeffers, K., Peak-Chew, S.Y., Raff, J.W. (2001).
Msps/XMAP215 interacts with the centrosomal protein D-TACC to regulate
microtubule behaviour. Nat. Cell. Biol. 3, 643–649.
72. Goshima, G., Wollman, R., Goodwin, S.S., Zhang, N., Scholey, J.M., Vale, R.D.,
Stuurman, N. (2007). Genes required for mitotic spindle assembly in Drosophila S2cells. Science 316, 417–421.
73. Sehadova, H., Glaser, F.T., Gentile, C., Simoni, A., Giesecke, A., Albert, J.T.,
Stanewsky, R. (2009). Temperature entrainment of Drosophila's circadian clock
involves the gene nocte and signaling from peripheral sensory tissues to the brain.
Neuron 64, 251–266.
74. Yoshikane, N., Nakamura, N., Ueda, R., Ueno, N., Yamanaka, S., Nakamura, M.
(2007). Drosophila NAT1, a homolog of the vertebrate translational regulator
NAT1/DAP5/p97, is required for embryonic germband extension and metamorphosis.
Dev. Growth Differ. 49, 623–634.
75. Cook, H.A., Koppetsch, B.S., Wu, J., Theurkauf, W.E. (2004). The Drosophila SDE3
homolog armitage is required for oskar mRNA silencing and embryonic axis
specification. Cell 116, 817–829.
76. Chen, Y., Pane, A., Schüpbach, T. (2007). Cutoff and aubergine mutations result in
retrotransposon upregulation and checkpoint activation in Drosophila. Curr. Biol. 17,
637–642.
77. Pane, A., Wehr, K., Schüpbach, T. (2007). zucchini and squash encode two putative
nucleases required for rasiRNA production in the Drosophila germline. Dev. Cell 12,
851–862.
78. Klattenhoff, C., Bratu, D.P., McGinnis-Schultz, N., Koppetsch, B.S., Cook, H.A.,
Theurkauf, W.E. (2007). Drosophila rasiRNA pathway mutations disrupt embryonicaxis specification through activation of an ATR/Chk2 DNA damage response. Dev
Cell 12, 45–55.
79. Hari, K.L., Santerre, A., Sekelsky, J.J., McKim, K.S., Boyd, J.B., Hawley, R.S. (1995).
The mei-41 gene of D. melanogaster is a structural and functional homolog of thehuman ataxia telangiectasia gene. Cell 82, 815–821.
80. Brodsky, M.H., Weinert, B.T., Tsang, G., Rong, Y.S., McGinnis, N.M., Golic, K.G.,
Rio, D.C., Rubin, G.M. (2004). Drosophila melanogaster MNK/Chk2 and p53
regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol.
Cell. Biol. 24, 1219–1231.
81. Klenov, M.S., Sokolova, O.A., Yakushev, E.Y., Stolyarenko, A.D., Mikhaleva, E.A.,
Lavrov, S.A., Gvozdev, V.A. (2011). Separation of stem cell maintenance andtransposon silencing functions of Piwi protein. Proc. Natl. Acad. Sci. USA. 108,
18760-18765.
82. Klenov, M.S., Lavrov, S.A., Korbut, A.P., Stolyarenko, A.D., Yakushev, E.Y., Reuter,
M., Pillai, R.S., Gvozdev, V.A. (2014). Impact of nuclear Piwi elimination on
chromatin state in Drosophila melanogaster ovaries. Nucleic. Acids Res. 42,
6208-6218.
83. Niki, Y., Yamaguchi, T., Mahowald, A.P. (2006). Establishment of stable cell lines ofDrosophila germ-line stem cells. Proc. Natl. Acad. Sci. USA. 103, 16325-16330.
84. Haase, A.D., Fenoglio, S., Muerdter, F., Guzzardo, P.M., Czech, B., Pappin, D.J.,
Chen, C., Gordon, A., Hannon, G.J. (2010). Probing the initiation and effector phasesof the somatic piRNA pathway in Drosophila. Genes Dev. 24, 2499-2504.
85. Matsumoto, N., Sato, K., Namba, Y., Miyakubi, K., Dohmae, N., Ishitani, R., Siomi, H.,
Nishimasu, H., Siomi, M.C., Nureki, O. (2015). Crystal structure and activity of theendoribonuclease domain of the piRNA pathway factor Maelstrom. Cell reports. In
press.
16

86. Chen, K., Campbell, E., Pandey, R.R., Yang, Z., Mccarthy, A., Pillai, R.S. (2015).
Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient
nuclease active in protists. RNA. In press.
87. Pace, C.N., Heinemann, U., Hahn, U., Saenger, W. (1991). Ribonuclease T1:
structure, function, and stability. Angew. Chem. Int. Ed. Engl. 30, 343–360.
88. Costa, Y., Speed, R.M., Gautier, P., Semple, C.A., Maratou, K., Turner, J.M., Cooke,
H.J. (2006). Mouse MAELSTROM: the link between meiotic silencing of unsynapsed
chromatin and microRNA pathway? Hum. Mol. Genet. 15, 2324-2334.
89. Soper, S.F., van der Heijden, G.W., Hardiman, T.C., Goodheart, M., Martin, S.L., deBoer, P., Bortvin, A. (2008). Mouse maelstrom, a component of nuage, is essential
for spermatogenesis and transposon repression in meiosis. Dev. Cell. 15, 285-297.
90. Aravin, A.A., van der Heijden, G.W., Castañeda, J., Vagin, V.V., Hannon, G.J.,
Bortvin, A. (2009). Cytoplasmic compartmentalization of the fetal piRNA pathway inmice. PLoS Genet. 5, e1000764.
91. Castañeda, J., Genzor, P., van der Heijden, G.W., Sarkeshik, A., Yates, J.R. 3rd,
Ingolia, N.T., Bortvin, A.(2014). Reduced pachytene piRNAs and translation underlie
spermiogenic arrest in Maelstrom mutant mice. EMBO J. 33, 1999-2019.
92. Pandey, R.R. & Pillai, R.S. (2014). Primary piRNA biogenesis: caught up in a
Maelstrom. EMBO J. 33, 1979-1980.
93. Vagin, V.V., Wohlschlegel, J., Qu, J., Jonsson, Z., Huang, X., Chuma, S., Girard, A.,
Sachidanandam, R., Hannon, G.J., Aravin, A.A. (2009). Proteomic analysis ofmurine Piwi proteins reveals a role for arginine methylation in specifying interaction
with Tudor family members. Genes Dev. 23, 1749–1762
94. Vasileva, A., Tiedau, D., Firooznia, A., Muller-Reichert. T., Jessberger, R. (2009).
Tdrd6 is required for spermiogenesis, chromatoid body architecture, and regulationof miRNA expression. Curr. Biol. 19, 630-639
95. Genzor, P. & Bortvin, A. (2015). A unique HMG-box domain of mouse Maelstrom
binds structured RNA but not double stranded DNA. bioRxiv.
96. Jiang, X., Huang, Q., Wang, W., Dong, H., Ly, H., Liang, Y., Dong, C. (2013).
Structures of arenaviral nucleoproteins with triphosphate dsRNA reveal a uniquemechanism of immune suppression. J. Biol. Chem. 288, 16949–16959.

17Figure legends
Figure 1. Crystal structure of the MAEL domain of Drosophila Mael.
Overall structures of Drosophila MAEL domain and a DEDDh exonuclease VASV NP[96]. The bound dsRNA is omitted for clarity. VASV NP has the DEDDh catalytic residues
in the catalytic groove, but Drosophila MAEL domain lacks them in the central groove.

Figure 2. Possible models of Mael function in piRNA biogenesis and transposon
silencing. Left: In Drosophila ovarian somatic cells, Mael is involved in Piwi-mediatedtranscriptional transposon silencing and acts downstream of H3K9me3 establishment by
Piwi. The MAEL domain, but not its ssRNase activity and HMG box, is indispensable forsilencing, which suggests that Mael may act as a scaffold of nuclear-effector complexes
to silence transposons. Right: In mouse adult testes, Mael interacts with Miwi and
TDRD6, and is required for pachytene piRNA biogenesis. During piRNA biogenesis,
Mael may bind to pachytene piRNA primary precursors through the HMG box andfragment it by the MAEL domain.

18

Figure 1̲Sato and Siomi
central groove
ECHC motif

catalytic groove

ECHC motif

Mn2+
Zn

2+

LASV NP(PDB 4GV9)

DmMAEL(PDB 4YBG)

Zn2+

Figure 2_sato and siomi

Mouse adult testes

Drosophila ovarian somatic cells
Transposons

H3K9me3

Mael

Pachyten piRNA cluster

Piwi
X
HMG-box(dispensable) MAEL domain(Scaffold?)

Pachyten piRNA
precursor

Nucleus
Cytoplasm
Mael complex
Piwi

Yb body

Nuage

MIWI TDRD6
HMG-box (Bindto precursors?)

MAEL domain(Fragmentation?)
Mael

MIWI