Report

Crystal Structure and Activity of theEndoribonuclease Domain of the piRNA Pathway
Factor Maelstrom
Graphical Abstract

Authors
Naoki Matsumoto, Kaoru Sato, ...,
Mikiko C. Siomi, Osamu Nureki

Correspondence
siomim@bs.s.u-tokyo.ac.jp (M.C.S.),
nureki@bs.s.u-tokyo.ac.jp (O.N.)

In Brief
Maelstrom (Mael) is essential fortransposon silencing in the piRNA
pathway. Using structural and functional
approaches, Matsumoto et al. show that
Mael lacks the canonical nuclease activesite structure but exhibits single-stranded
RNA cleavage activity, unrelated totransposon silencing, in flies.

Highlightsd

Crystal structure of the MAEL domain in Drosophila
Maelstrom is determinedd

The MAEL domain has an RNase H-like fold but lacks
canonical catalytic residuesd

The MAEL domain shows single-strand-specificendoribonuclease activityd

The ssRNase activity of Mael is unrelated to transposon
silencing

Matsumoto et al., 2015, Cell Reports 11, 366–375
April 21, 2015 ª2015 The Authors
http://dx.doi.org/10.1016/j.celrep.2015.03.030

Accession Numbers
4YBG
LC032360

Cell Reports

ReportCrystal Structure and Activityof the Endoribonuclease Domainof the piRNA Pathway Factor Maelstrom
Naoki Matsumoto,1,5 Kaoru Sato,1,5 Hiroshi Nishimasu,1,2,5 Yurika Namba,1 Kana Miyakubi,1 Naoshi Dohmae,3Ryuichiro Ishitani,1 Haruhiko Siomi,4 Mikiko C. Siomi,1,* and Osamu Nureki1,*
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
2JST, PRESTO, Tokyo 113-0032, Japan
3Biomolecular Characterization Team and CREST/JST, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
4Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
5Co-first author*Correspondence: siomim@bs.s.u-tokyo.ac.jp (M.C.S.), nureki@bs.s.u-tokyo.ac.jp (O.N.)
http://dx.doi.org/10.1016/j.celrep.2015.03.030
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARYPIWI-interacting RNAs (piRNAs) protect the genome
from transposons in animal gonads. Maelstrom(Mael) is an evolutionarily conserved protein, composed of a high-mobility group (HMG) domain and a
MAEL domain, and is essential for piRNA-mediatedtranscriptional transposon silencing in various species, such as Drosophila and mice. However, its
structure and biochemical function have remained
elusive. Here, we report the crystal structure of the
MAEL domain from Drosophila melanogaster Mael,
at 1.6 Å resolution. The structure reveals that the
MAEL domain has an RNase H-like fold but lacks canonical catalytic residues conserved among RNase
H-like superfamily nucleases. Our biochemical analyses reveal that the MAEL domain exhibits singlestranded RNA (ssRNA)-specific endonuclease activity. Our cell-based analyses further indicate thatssRNA cleavage activity appears dispensable forpiRNA-mediated transcriptional transposon silencing
in Drosophila. Our findings provide clues toward understanding the multiple roles of Mael in the piRNA
pathway.
INTRODUCTION
Small RNA-based defense systems repress the aberrant expression of transposable elements (TEs) and thus maintain genome
integrity in animal gonads (Malone and Hannon, 2009; Siomi
et al., 2011). The germline-specific PIWI clade of Argonaute family proteins and the 23- to 30-nt noncoding PIWI-interacting
RNAs (piRNAs) are the core of this defense system. PIWI proteins bind piRNAs to form piRNA-induced silencing complexes(piRISCs), which silence their complementary target TEs at thetranscriptional or posttranscriptional level (Malone and Hannon,
2009; Siomi et al., 2011; Ishizu et al., 2012; Luteijn and Ketting,
366 Cell Reports 11, 366–375, April 21, 2015 ª2015 The Authors

2013). The Drosophila genome encodes three PIWI proteins:
Piwi, Aubergine (Aub), and Argonaute3 (AGO3). The Drosophila
ovary consists of two types of cells, somatic cells such as follicle
cells, and germ cells such as nurse cells and oocytes. Piwi islocalized in the nucleus in both somatic and germ cells, where
it participates in the primary piRNA pathway (Czech et al.,2013; Handler et al., 2013; Olivieri et al., 2010). In contrast, Aub
and AGO3 are enriched in cytoplasmic perinuclear granules
called nuage in germ cells, where they participate in secondary
piRNA biogenesis (Brennecke et al., 2007; Gunawardane et al.,2007; Li et al., 2009; Malone et al., 2009). In the primary piRNA
pathway in Drosophila ovarian somatic cells, single-stranded,long piRNA precursors are transcribed from discrete genomic
loci, called piRNA clusters, and are processed into mature piRNAs by the single-strand-specific endoribonuclease Zucchini(Zuc) (Ipsaro et al., 2012; Nishimasu et al., 2012). The primarypiRNAs are loaded into Piwi at cytoplasmic perinuclear Yb
bodies (Saito et al., 2010). piRISC then enters the nucleus
and promotes repressive histone H3 lysine 9 trimethylation(H3K9me3), thereby silencing target TEs at the transcriptional
level (Sienski et al., 2012; Wang and Elgin, 2011; Le Thomas
et al., 2013; Rozhkov et al., 2013). In the secondary piRNAbiogenesis pathway, Aub and AGO3 reciprocally cleave sense
and antisense TE transcripts, respectively (Brennecke et al.,2007; Gunawardane et al., 2007). This feed-forward piRNA
amplification loop, called the ping-pong cycle, enables simultaneous secondary piRNA biogenesis and TE silencing.
Maelstrom (Mael) is an evolutionarily conserved protein implicated in the piRNA pathway (Lim and Kai, 2007; Soper et al.,2008; Aravin et al., 2009; Sienski et al., 2012; Castañeda et al.,2014). In somatic cells of the fly ovary, Mael is predominantly
localized in the nucleus (Sienski et al., 2012). In contrast, ingerm cells of the fly ovary and mouse testis, Mael is localized
in both the nucleus and cytoplasmic granules (nuage in flies,
piP-bodies or chromatoid bodies in mice) (Findley et al., 2003;
Costa et al., 2006; Lim and Kai, 2007; Soper et al., 2008; Aravin
et al., 2009; Sato et al., 2011; Castañeda et al., 2014). Mael isalso implicated in various biological processes, such as oocyte
development and germline stem cell (GSC) differentiation (Clegg

et al., 1997, 2001; Pek et al., 2009, 2012; Sato et al., 2011).
Mael is composed of an N-terminal HMG domain and a central
MAEL domain, which was predicted to adopt an RNase H-likefold by a bioinformatics analysis (Zhang et al., 2008) (Figures
1A and S1). However, the biochemical function of Mael remains
elusive.
In cultured Drosophila ovarian somatic cells (OSCs) (Niki et al.,2006; Saito et al., 2009), mael knockdown (KD) did not affectpiRNA biogenesis but resulted in the derepression of TEs, indicating that Mael is essential for Piwi-mediated TE silencing(Sienski et al., 2012). Intriguingly, mael KD only modestly
impacted the H3K9me3 patterns at the target heterochromaticloci, suggesting that Mael acts downstream of or in parallel tothe Piwi-mediated H3K9me3 modification (Sienski et al., 2012).
In addition, Mael has been implicated in piRNA biogenesis ingerm cells of the fly ovary and mouse testis (Lim and Kai,
2007; Sienski et al., 2012; Aravin et al., 2009; Castañeda et al.,2014). Despite the crucial role of Mael in the piRNA pathway,
the molecular mechanism of Mael/Piwi-mediated TE silencing
remains elusive, due to the lack of structural and biochemical
information.
In this study, we solved the crystal structure of the MAEL
domain of D. melanogaster Mael. The structure revealed that
the MAEL domain adopts an RNase H-like fold but lacks the canonical catalytic residues conserved among the RNase H-likesuperfamily of endonucleases and exonucleases. Moreover,
our biochemical and biological analyses revealed that the
MAEL domain has single-stranded RNA (ssRNA) cleavage activity, which appears dispensable for Mael/Piwi-mediated transcriptional TE silencing in Drosophila OSCs. Our findings provide
clues toward understanding the multiple functions of Mael in thepiRNA pathway.
RESULTS
Crystal Structure of the MAEL Domain from
D. melanogaster Mael
To gain mechanistic insights into the function of Mael, we
attempted to determine the crystal structure of full-length
D. melanogaster Mael (residues 1–459, referred to as FLDmMael) but were hampered by its low expression levels inEscherichia coli. Limited trypsin proteolysis of FL-DmMael revealed that the MAEL domain (residues 84–333, referred to as
DmMAEL) is a well-expressed, stable region suitable for structural analysis (Figure 1A). Furthermore, we found that the substitution of a less-conserved cysteine residue (Cys228) with serine
dramatically improved the diffraction quality. X-ray fluorescence
spectra of the crystal indicated that DmMAEL binds a zinc ion,
consistent with a previous bioinformatics analysis suggestingthat a zinc ion is coordinated by the ECHC motif, which isconserved among Mael orthologs (Zhang et al., 2008) (Figures
S1 and S2). We determined the crystal structure of DmMAEL(C228S) at 1.6 Å resolution by the single-wavelength anomalous
diffraction (SAD) method, using the intrinsic zinc atom (Figure 1B;
Table S1). The structure revealed that DmMAEL consists of atwisted five-stranded mixed b sheet surrounded by 13 helices,with a zinc ion coordinated by Glu131, Cys288, His291, andCys300 in the ECHC motif (Figure 1C).

A Dali search (Holm and Rosenström, 2010) revealed that
DmMAEL shares structural similarity with the RNase H-like superfamily of endonucleases and exonucleases (Majorek et al.,2014), especially with the DEDDh family exonucleases, such asa Lassa virus nucleoprotein (LASV NP) (PDB 4GV9) (Jiang
et al., 2013). LASV NP is a 30 –50 exonuclease involved in the
suppression of virus-induced interferon production (Martı́nezSobrido et al., 2007; Qi et al., 2010; Hastie et al., 2011; Jiang
et al., 2013). Despite their low sequence identity (13%),
DmMAEL shares an RNase H-like fold, consisting of a fivestranded b sheet flanked by a helices on both sides, with LASVNP (Jiang et al., 2013) (root mean square deviation of 2.9 Å for
131 aligned Ca atoms) (Figure 1D). Like DmMAEL, LASV NP contains a zinc ion coordinated by the ECHC motif, consisting ofGlu399, Cys506, His509, and Cys529, which may contribute to
structural stabilization or substrate binding (Qi et al., 2010; Hastie et al., 2011) (Figures 1E and S3A). In DmMAEL, the bound zincion may play at least a structural role, since point mutations of the
ECHC motif drastically reduced the solubility of DmMAEL in vitro(data not shown). The DEDDh family exonucleases have a negatively charged catalytic groove formed by five invariant catalytic
residues (Asp, Glu, Asp, Asp, and His; DEDDh motif) andcleave double-stranded RNAs (dsRNAs) through a two-metalion mechanism (Zuo and Deutscher, 2001). In LASV NP, the catalytic groove is formed by Asp389, Glu391, Asp466, Asp533,
and His528 in the DEDDh motif and the highly conserved
Ser430, Gln462, and Arg492 residues (Hastie et al., 2012; Jiang
et al., 2013) (Figures 1F and S3B). The Asp389, Glu391, Asp466,
Asp533, and His528 residues of LASV NP respectively correspond to the Ala114, Asn116, Met218, Met304, and Tyr299
residues of DmMAEL (Figures 1F and S3B). Consequently, thecentral groove of DmMAEL, which corresponds to the catalytic
groove of LASV NP, is not negatively charged (Figure 1G). In
addition, the residues in the central groove are not conserved
among Mael orthologs (Figures S1 and S2), suggesting that thecentral groove is less important for the function of Mael. Taken
together, the crystal structure of DmMAEL revealed that it
adopts an RNase H-like fold but lacks the canonical catalytic
residues conserved among RNase H-like superfamily members.
The MAEL Domain Has Single-Strand-SpecificEndoribonuclease Activity
To determine whether DmMAEL is a nuclease, we measured
the nuclease activity of purified DmMAEL using a 50 32P-labeled
40-nt ssRNA (40AS ssRNA) as the substrate. Unexpectedly,
DmMAEL cleaved the 40AS ssRNA (Figure 2A). The elution
profile of purified DmMAEL correlated closely with that of thesingle-stranded ribonuclease (ssRNase) activity in gel-filtration
chromatography (Figure 2A). Furthermore, DmMAEL cleaved
the 40AS ssRNA in a dose- and time-dependent manner (Figures
2B and 2C). These results revealed that DmMAEL is a ssRNase.
The ssRNase activity of DmMAEL did not require divalent metal
ions, such as Mg2+ or Ca2+, and was rather inhibited in theirpresence (Figure 2D). These results are consistent with our
structural finding that DmMAEL shares no catalytic residues
with the DEDDh family members, which require divalent metal
ions, such as Mg2+ and Mn2+, for substrate cleavage (Zuo andDeutscher, 2001). To determine the substrate specificity ofCell Reports 11, 366–375, April 21, 2015 ª2015 The Authors 367

A

C
HMG
1

MAEL
71 84

333

His291459

DmMAEL

Glu131B
Cys300α2

Zn2+

ECHC motif

Cys288α4α5η2N (84)
β5

α3

β1 β2

η3

D

α8

dsRNA

α9

β4
α7

β3

Zn2+

Zn2+

Mn2+

C (332)α1
α6LASV NP
η1

η4
DmMAELE

F
Cys288Cys506

Thr171

His291
His509

Asn116

Ser430Glu391

Tyr299

Met218His528

Asp466

Zn2+
Zn2+

Cys300Cys529

Mn2+

Asn215
Arg307
Ala114

Asp389
Met304

Asp533Thr211

DmMAELLASV NP

Glu131Glu399Gln462

Pro212
Trp308LASV NPECHC motif

ECHC motif
dsRNA

180˚

dsRNA

180˚

Catalytic groove

Central groove

Arg492

Asp278DmMAELG

Mn2+

LASV NP

DmMAEL(legend on next page)368 Cell Reports 11, 366–375, April 21, 2015 ª2015 The Authors

DmMAEL, we next measured the cleavage activity toward a
series of 50 32P-labeled nucleic acid substrates. DmMAEL
efficiently cleaved ssRNA, but neither dsRNA nor ssDNA (Figure 2E). DmMAEL cleaved circular 40AS ssRNA, indicating that
DmMAEL is an endoribonuclease (Figure 2F). The cleavage
pattern of the 40AS ssRNA revealed that DmMAEL preferentiallycleaves ssRNA at a guanine residue (especially at successive
guanine stretches) (Figure 2E). To exclude the possibility thatthe 40AS ssRNA adopts a secondary structure that affects thecleavage by DmMAEL, we measured the nuclease activity of
DmMAEL toward 15-nt poly(A) RNA substrates with or without
guanine residues, which are unlikely to adopt secondary structures. DmMAEL cleaved 15-nt poly(A) containing guanine residues, but not 15-nt poly(A), confirming that DmMAEL cleaves
ssRNA at guanine residues (Figure 2G). We next compared thecleavage patterns of the 40AS ssRNA by DmMAEL and RNase
T1, an endonuclease that specifically cleaves ssRNA at the 30
side of guanine residues (Pace et al., 1991). RNase T1 cleaved
the 40AS ssRNA evenly at guanine residues (Figure 2H). In
contrast, DmMAEL did not efficiently cleave the 40AS ssRNA
at 3 nt from the 50 end (position 1) and 4 nt from the 30 end (position 6) (Figure 2H). The ssRNase activity of DmMAEL was inhibited in the presence of 25 mM NaCl, whereas that of RNase
T1 remained robust in the presence of 100 mM NaCl (Figure 2I).
These differences in their enzymatic properties indicated that the
RNA cleavage mechanism of DmMAEL is distinct from that of
RNase T1. To examine whether the nuclease activity is specificto D. melanogaster Mael, we measured the ssRNase activitiesof the purified MAEL domains from Bombyx mori Mael (residues
92–335, referred to as BmMAEL) and Mus musculus Mael (residues 83–327, referred to as MmMAEL) (Figure 2J). We foundthat both BmMAEL and MmMAEL cleave the 40AS ssRNA in
similar manners to that of DmMAEL, although the ssRNase
activity of MmMAEL was weaker than those of DmMAEL and
BmMAEL (Figure 2J). Together, these biochemical data revealed
that the MAEL domain is an evolutionarily conserved, singlestrand-specific endoribonuclease.
Potential RNA-Binding Residues of the MAEL Domain
Since the MAEL domain lacks the canonical DEDDh motif, we
tried to identify the catalytic residues of DmMAEL, based on thesequence conservation among Mael orthologs. However, multiple sequence alignments indicated that only the ECHC motif issolvent accessible and strictly conserved across the Mael ortho-

logs (Figures S1 and S2). Thus, based on the crystal structure of
DmMAEL, we prepared 12 DmMAEL mutants, in which the solvent-exposed, hydrophilic residues were individually substituted
with alanine (Figure 3A). All of the mutants eluted as a single
monodisperse peak from the gel-filtration column (data notshown), confirming their structural integrity. We then examined
the ssRNase activities of the purified mutants, using 40AS ssRNA
as the substrate (Figure 3B). The K109A, K188A, N192A, andE292A mutants showed ssRNase activities comparable to thatof the wild-type DmMAEL, and the K277A mutant showed
moderately reduced ssRNase activity (Figure 3B). In contrast,
the K140A, K199A, Q289A, D293A, D295A, D314A, and K328A
mutants showed markedly reduced ssRNase activities (Figure 3B), indicating that Lys140, Lys199, Gln289, Asp293,
Asp295, Asp314, and Lys328 are involved in the ssRNase activity. Although DmMAEL, BmMAEL, and MmMAEL cleaved ssRNAin a similar manner (Figure 2J), these residues (except for Asp295)
are not conserved among the Mael orthologs (Figure S1). Moreover, these mutations reduced, but did not abolish, the ssRNase
activity (Figure 3B), suggesting that these residues are involved inssRNA binding, but not in catalysis. The positively charged residues, Lys140, Lys199, and Lys328, would interact with the negatively charged phosphate backbone of the ssRNA substrates.
These residues are located on the opposite side of the central
groove, which is equivalent to the catalytic groove of LASV NP(Figures 3C and 3D). To examine whether the central groove isinvolved in the ssRNase activity, we tried to prepare four additional DmMAEL mutants (N116A, M218A, Y299A, and M304A),
in which the residues corresponding to the DEDDh motif wereindividually substituted with alanine. These four mutants were
not expressed in E. coli as soluble proteins (data not shown), suggesting that these residues within the central groove contribute to
structural integrity. In addition, the C228S mutant used for our
structural analysis exhibited the ssRNase activity (Figure 3B),
indicating that the C228S mutation does not have considerable
impact on the structure and function of DmMAEL.
Since both DmMAEL and RNase T1 preferentially cleave ssRNA
at guanine residues, we attempted to detect the structural similarity between them. RNase T1 cleaves ssRNA through a metal-ionindependent mechanism, in which a conserved histidine serves asa catalytic residue (Pace et al., 1991). Since His329 of DmMAEL is
the only histidine residue conserved among DmMAEL, BmMAEL,
and MmMAEL (Figure S1), we examined the RNase activity of the
DmMAEL H329A mutant (Figure 3A). The H329A mutant retained

Figure 1. Crystal Structure of DmMAEL(A) Domain structure of D. melanogaster Mael.
(B) Overall structure of DmMAEL. The zinc ion is shown as a gray sphere. Disordered regions (residues 156–162, 228–229, and 236–237) are shown as dashedlines.
(C) Close-up view of the ECHC motif. An FO  FC simulated annealing omit map contoured at 3.5s is shown as a blue mesh.
(D) Structures of DmMAEL (left) and LASV NP in complex with dsRNA (PDB ID 4GV9) (right). The zinc ions are shown as light blue and yellow spheres in DmMAEL
and LASV NP, respectively. The manganese ions are shown as pink spheres. The ECHC motif and the central groove are indicated by green and red boxes,
respectively.
(E) Superimposition of the ECHC motifs of DmMAEL and LASV NP.
(F) Central grooves of DmMAEL (left) and LASV NP (right). In LASV NP, the DEDDh-motif and RNA-binding residues are shown as magenta and white sticks,
respectively. The bound manganese ions are shown as pink spheres, and the bound dsRNA is omitted for clarity. In DmMAEL, the equivalent residues in thecentral groove are shown in the same color code. Coordination bonds are shown as dashed lines.
(G) Electrostatic surface potentials of DmMAEL and LASV NP (contoured from 5 kT/e [red] to +5 kT/e [blue]).
See also Figures S1–S3 and Table S1.

Cell Reports 11, 366–375, April 21, 2015 ª2015 The Authors 369

Absorbance at 280 nm

C

D

DmMAEL (μM)

DmMAEL

Metal ion

Time (min)

(nt)
50
40
30
10

15

20

180
15
30
60
120
180

B

Fraction
1 2345678

0.14
0.28
0.55
1.1
2.2

A

-

DmMAEL +

(nt)

(nt)

50
40

50
40

30

Mg2+

Ca2+

-

-

+

+

30

24

Elution volume (ml)

20

20
20

(kDa)
250
150
100
75
50
37

40AS ssRNA

40AS ssRNA

F

E

10

Substrate ssRNA ssRNA dsRNA ssDNA ssDNA
S
AS
S/AS
S
AS
2

3

4

5

6

7

DmMAEL

8

- +

- +

- +

- +

Linear Circular
DmMAEL

15

Fraction 1

40AS ssRNA

- +

(nt)

(nt)

(nt)

40

60
50

50

40

40

30

30

30

20

G
15-nt poly(A)

ExoT

25
20

0G

1G

3G

DmMAEL - + - + - +
(nt)

40
30

20
20

20

10

*
40AS ssRNA

40AS ssRNA

(nt)
50
40

6

30

5
4

20

3

2
1
40AS ssRNAJ
No enzyme

1

NaCl

DmMAEL
NaCl

RNase T1
NaCl(mM) 0 25 50 100 0 25 50 100 0 25 50 100
(nt)

2
50
40

3

-

(kDa)
250
150
100
75

(nt)

50

30

30

DmMAEL
BmMAEL
MmMAEL

RNase T1I

DmMAEL
BmMAEL
MmMAELDmMAEL

5′-CUGCCACUUUUGUUCCAGGGAACCAGAUCGAAAUCAGACC-3′

H

40

37
20

4

20

25
20

5
10
15

6
10

40AS ssRNA

40AS ssRNA(legend on next page)370 Cell Reports 11, 366–375, April 21, 2015 ª2015 The Authors

the ssRNase activity, indicating that His329 is not involved inssRNA cleavage (Figure 3B). Thus, despite our numerous attempts, the RNA cleavage mechanism remains to be elucidated.
Nonetheless, our mutational analyses support the notion that
Mael does not share an active site with either RNase T1 or the
DEDDh family members.
The ssRNase Activity of Mael Appears Dispensable for
Piwi/Mael-Mediated TE Silencing in Drosophila OSCs
To examine the contribution of the MAEL domain to Piwi/
Mael-mediated TE silencing, we overexpressed DmMAEL,
FL-DmMael, the four ECHC-motif single mutants (E131A,
C288A, H291A, and C300A) of FL-DmMael, the ECHC-motif
quadruple mutants (E131A/C288A/H291A/C300A) of FLDmMael, and DmMAEL in mael-depleted OSCs and then monitored the expression levels of TEs by qRT-PCR. FL-DmMael and
DmMAEL rescued the derepression of a variety of somatic TEs(mdg1, 297, blood, Tabor, gypsy, and ZAM) (Figures 4A, 4B,
and S4A), indicating that the MAEL domain plays a central
role in Piwi/Mael-mediated TE silencing in Drosophila OSCs,
consistent with a previous report (Sienski et al., 2012). All of
the ECHC-motif mutants of FL-DmMael and DmMAEL failed
to rescue TE derepression (Figures 4A and S4B), highlighting
the functional significance of the ECHC motif for Piwi/Maelmediated TE silencing.
To examine whether the ssRNase activity of DmMAEL is
involved in Piwi/Mael-mediated TE silencing, we next overexpressed the seven ssRNase-deficient DmMAEL mutants(K140A, K199A, Q289A, D293A, D295A, D314A, and K328A) in
mael-depleted OSCs and then monitored the levels of TE derepression. Like wild-type DmMAEL, all seven of the ssRNasedeficient mutants rescued TE derepression (Figures 4A and
S4C). Since the ECHC-motif mutants of DmMAEL were not expressed in E. coli as soluble proteins, we could not examine their
ssRNase activities in vitro. Overall, these results suggested thatthe ssRNA cleavage activity of Mael appears dispensable for
Piwi/Mael-mediated TE silencing in Drosophila OSCs.
DISCUSSION
The present crystal structure revealed that DmMAEL adopts
an RNase H-like fold but does not share catalytic residues

with the RNase H-like superfamily members. Unexpectedly,
our biochemical analyses revealed that DmMAEL has ssRNase
activity. We further showed that BmMAEL and MmMAEL also
have the ssRNase activities, and we identified seven potential
ssRNA-binding residues of DmMAEL. These results strongly
support our surprising finding that the MAEL domain possesses the ssRNase activity, although it lacks the canonicalactive site conserved across the RNase H-like superfamily
nucleases.
Previous studies showed that Mael participates in piRNAbiogenesis in germ cells of the fly ovary and mouse testis (Limand Kai, 2007; Sienski et al., 2012; Aravin et al., 2009; Castañeda
et al., 2014). In the adult mouse testis, a ribonucleoprotein complex comprising Mael, the PIWI protein MIWI, and the Tudordomain-containing protein TDRD6 is involved in the processingof precursor transcripts into mature pachytene piRNAs, a classof mammalian piRNAs (Castañeda et al., 2014). Notably, thenuclease activity of MIWI is not required for piRNA biogenesis(Reuter et al., 2011), and the ribonucleoprotein complex lacks apotential nuclease (Castañeda et al., 2014), suggesting that
Mael may be responsible for the processing of pachytene piRNA
precursors in adult mouse testis. This model is consistent with ourfinding that MmMAEL exhibits the ssRNase activity. Together,
these observations suggested that the ssRNase activity of Maelis involved in the processing of piRNA precursors in mice.
A previous study showed that the MAEL domain plays a central role in Piwi/Mael-mediated TE silencing in Drosophila OSCs
(Sienski et al., 2012). In OSCs, the mael KD has mild effects on
the establishment of H3K9me3 but increases RNA polymerase
II occupancy at target heterochromatic loci, thereby resulting in
the derepression of TEs (Sienski et al., 2012). These observations indicated that Mael acts downstream of or in parallel tothe H3K9me3 modification event. A large-scale genetic screen
further indicated that, in addition to Piwi and Mael, the zincfinger domain-containing protein Gtsf1 (Dönertas et al., 2013;
Ohtani et al., 2013) and several chromatin-associated factors,
such as the histone deacetylase HDAC3 and the histone chaperone Asf1, are involved in the Drosophila somatic piRNA
pathway (Handler et al., 2013; Muerdter et al., 2013). Consistent with the previous report (Sienski et al., 2012), our cellbased analysis indicated that the MAEL domain is involved in
TE silencing in Drosophila OSCs. Our mutational analysis

Figure 2. ssRNA-Cleavage Activity of the MAEL Domain(A) ssRNase activity of DmMAEL. The gel-filtration chromatography elution profile of DmMAEL (top). SDS-PAGE analysis (middle) and ssRNase activity offractions 1–8 (bottom). Each fraction (1 ml) was incubated with the 40AS ssRNA and then analyzed by 15% denaturing PAGE.
(B) Dose dependency of the DmMAEL ssRNase activity. DmMAEL (0.14–2.2 mM) was incubated with the 40AS ssRNA at 26 C for 3 hr.
(C) Time dependency of the DmMAEL ssRNase activity. DmMAEL (2.2 mM) was incubated with the 40AS ssRNA at 26 C for 15–180 min.
(D) Effect of divalent cations on the DmMAEL ssRNase activity.
(E) Substrate specificities of DmMAEL. ‘‘S’’ and ‘‘AS’’ indicate the sense and antisense strands of dsRNA, respectively.
(F) ssRNase activity of DmMAEL toward linear and circular 40AS ssRNA. Exonuclease T (ExoT) was used as a control.
(G) ssRNase activity of DmMAEL toward 15-nt poly(A) ssRNA with and without guanine residues. The asterisk indicates the cleavage products.
(H) Comparison of the ssRNase activities of DmMAEL with RNase T1. DmMAEL (0.14–2.2 mM) or RNase T1 (0.5–10 units) was incubated with the 40AS ssRNA at
26 C for 3 hr. The nucleotide sequence of the substrate 40AS ssRNA is shown on the right of the gel, with guanine residues highlighted in bold. The predictedcleavage sites are indicated by red numbers.
(I) Comparison of the ssRNase activities of DmMAEL and RNase T1 in the presence of NaCl.
(J) ssRNase activities of DmMAEL, BmMAEL, and MmMAEL. SDS-PAGE analysis (left) and ssRNase activities (right) of purified DmMAEL, BmMAEL, and
MmMAEL.
See also Table S2.

Cell Reports 11, 366–375, April 21, 2015 ª2015 The Authors 371

WT
K109A
K140A
K188A
N192A
K199A
K277A
Q289A
E292A
D293A
D295A
D314A
K328A
H329A
C228S

A

C
Central groove

(kDa)
250
150
100
75

ECHC motif
Asp293
Asp295Gln289Lys188

Asp293
Asp295Gln289 Glu292

50
37

Asn192

Glu292
25
20

Lys277115˚
Asp31415

Lys199

10
Lys277
Lys109

B
-

WT
K109A
K140A
K188A
N192A
K199A
K277A
Q289A
E292A
D293A
D295A
D314A
K328A
H329A
C228S

Asp314

Lys328
Lys140
His329D
Central groove

ECHC motif(nt)

Asp293
Asp29550
40

Gln289Lys188

Asp293
Asp295Gln289 Glu292

30

Asn192

Glu29220

Lys277115˚
Asp314
Lys199Lys277
Asp31440AS ssRNA

Lys109Lys328
Lys140
His329Figure 3. Potential ssRNA-Binding Residues of DmMAEL(A) Purified wild-type and mutants of DmMAEL. WT, wild-type.
(B) ssRNase activities of purified wild-type and mutants of DmMAEL. The ssRNase activities were measured using the 40AS ssRNA as the substrate.
(C) Structural mapping of the ssRNase-deficient mutations. Residues involved in ssRNA cleavage are colored red, whereas residues not involved in ssRNA
cleavage are colored cyan. The ECHC motif is colored yellow. The central groove is indicated by a gray circle.
(D) Electrostatic surface potentials of DmMAEL, viewed from the same direction as in (C) (contoured from 5 kT/e [red] to +5 kT/e [blue]).

further suggested that the ssRNase activity of the MAEL
domain appears dispensable for TE silencing. Thus, we propose that Mael interacts with other piRNA factors via the
MAEL domain and thereby participates in TE silencing in
Drosophila OSCs.
A previous bioinformatics analysis suggested that the MAEL
domain evolved from a DEDDh exonuclease by switching thecatalytic residues from the DEDDh motif to the ECHC motif
and that the MAEL domain may possess the nuclease activity(Zhang et al., 2008). Consistent with this, our structural andbiochemical data revealed that the MAEL domain lacks the
DEDDh motif but shows ssRNase activity. Given that the
ECHC motif is strictly conserved among Mael orthologs (Figures
S1 and S2), the ECHC motif may play a catalytic role in addition
to a structural role. This idea is supported by the observation that
Asp295 of DmMAEL, which is highly conserved and locatedclose to the ECHC motif, is involved in the ssRNase activity.
372 Cell Reports 11, 366–375, April 21, 2015 ª2015 The Authors

If the ECHC motif participates in catalysis, then it is possiblethat the ssRNase activity of Mael is involved in TE silencing in
Drosophila OSCs, since the ECHC-motif mutants failed to rescue
TE derepression in our cell-based rescue experiments. All of
the ssRNase-deficient DmMAEL mutants we examined in ourcell-based assays retained slight ssRNase activities in vitro,
which might be sufficient for TE silencing when overexpressedin OSCs. Indeed, in our previous study on Zuc, an endoribonuclease implicated in primary piRNA biogenesis, TE derepression
was not rescued by the overexpression of the catalyticallyinactive Zuc mutant but was efficiently rescued by the overexpression of the RNA-binding-deficient Zuc mutants retaining residual ssRNase activity (Nishimasu et al., 2012). Thus, we cannot
completely rule out the possibility that the ssRNase activity ofMael is required for TE silencing. To fully understand the multiple
roles of Mael, an enigmatic key factor in the piRNA pathway, it
will be critical to elucidate (1) its ssRNA cleavage mechanism,

1

1

1

0

0

1

EGFP
FL-DmMael
DmMAEL

EGFP
WT
ECHC
D295A
D314A

0

0

DmMAELFL-DmMael

100(A) Repression of the mdg1 transposon by FLDmMael, DmMAEL, the ECHC-motif mutants of
FL-DmMael, the ECHC-motif quadruple mutant
of DmMAEL, and the ssRNase-deficient mutantsof DmMAEL. Myc-tagged proteins were overexpressed in mael-depleted Drosophila OSCs, andthe expression levels of the mdg1 transposon weremonitored by qRT-PCR (n = 3; error bars indicate
SEM). Myc-tagged EGFP was used as a control.
The ECHC mutant represents the E131A/C288A/
H291A/C300A quadruple mutant.
(B) Repression of a subset of TEs by FL-DmMaeland DmMAEL.
See also Figure S4 and Table S2.

DmMAEL

mdg1
297
blood
Tabor
gypsy
ZAM
HeT-A

80

Relative expression

Figure 4. Effects of Mael Mutations on TE
Silencing in OSCs

2

2

Rescueplasmid

B

2

3

EGFP
WT
ECHC
K140A
K199A
Q289A
D293A
K328A

Relative mdg1 expression

2

EGFP
WT
ECHC
E131A
C288A
H291A
C300A

A

60

40

20

0

Rescueplasmid

EGFP

FL-DmMael

DmMAEL(2) its endogenous ssRNA substrates, and (3) the physiologicalrelevance of its ssRNase activity.

mori Mael have been deposited in GenBank under accession number
LC032360.

EXPERIMENTAL PROCEDURES

SUPPLEMENTAL INFORMATION

Detailed experimental procedures are described in Supplemental Experimental Procedures, and related sequences are shown in Table S2.
DmMAEL (residues 84–333, C228S) was expressed in E. coli as a
His-tagged protein, and purified by chromatography on Ni-NTA Superflow(QIAGEN) and Resource Q (GE Healthcare) columns. Crystals were obtained
at 20 C by the sitting-drop vapor diffusion method. X-ray diffraction data
were collected on beamline BL32XU at SPring-8 (Hyogo). The crystal structure of DmMAEL was determined by the SAD method, using the intrinsic
zinc atom.
For nuclease activity measurements, the wild-type and mutants of DmMAEL
and BmMAEL (residues 92–335) were expressed in E. coli as His-tagged proteins, and the proteins were purified by chromatography on Ni-NTA Superflow,
Resource Q, Resource PHE (GE Healthcare), and Superdex 200 10/300 (GEHealthcare) columns. MmMAEL (residues 83–327) was expressed in Sf9 insect
cells as a His-SUMOstar-tagged protein (LifeSensors) and purified by chromatography using a similar protocol as for DmMAEL. Nuclease activity measurements were performed in buffer containing 25 mM HEPES-KOH (pH 7.4) and5 mM DTT. Rescue experiments were performed essentially as described previously (Nishimasu et al., 2012).

Supplemental Information includes Supplemental Experimental Procedures,four figures, and two tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2015.03.030.

ACCESSION NUMBERS
The atomic coordinates of DmMAEL have been deposited in the Protein
Data Bank under accession number 4YBG. The sequencing data of Bombyx

AUTHOR CONTRIBUTIONS
N.M., K.S., H.S., H.N., M.C.S., and O.N. designed the experiments; N.M., R.I.,
and H.N. performed the structural analysis; N.M. performed the nuclease activity measurement; K.S., Y.N. and K.M. performed the cell-based analysis;
N.D. performed the mass spectrometric analysis; N.M., K.S., H.N., M.C.S.,
and O.N. wrote the manuscript; and all authors discussed the data and themanuscript. H.N., M.C.S., and O.N. supervised all of the work.
ACKNOWLEDGMENTS
We thank Arisa Kurabayashi for technical assistance, Tomoya Tsukazaki and
Kaoru Kumazaki for assistance with data collection, and the beamline staff at
BL32XU of SPring-8 for technical assistance during data collection. The X-ray
diffraction experiments were performed at BL32XU of SPring-8 (proposal no.
2011B1280). This work was supported by a grant from the Target Protein
Research Program (TPRP) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to O.N.), by a grant from the Core Research for
Evolutional Science and Technology (CREST) Program, The Creation of Basic
Chronic Inflammation from Japan Science and Technology Agency (JST) (toCell Reports 11, 366–375, April 21, 2015 ª2015 The Authors 373

O.N.), and by a Grant-in-Aid for Scientific Research from MEXT (to H.N. and
M.C.S.).
Received: October 17, 2014
Revised: February 19, 2015
Accepted: March 10, 2015
Published: April 9, 2015
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Cell Reports
Supplemental Information

Crystal Structure and Activityof the Endoribonuclease Domainof the piRNA Pathway Factor Maelstrom
Naoki Matsumoto, Kaoru Sato, Hiroshi Nishimasu, Yurika Namba, Kana Miyakubi,
Naoshi Dohmae, Ryuichiro Ishitani, Haruhiko Siomi, Mikiko C. Siomi, and Osamu
Nureki

HMG domain
1

Dm
Bm
Xt
Mm
Hs

10

20

30

40

α1
70

Dm
Bm
Xt
Mm
Hs

80

η1

90

100

β1

β2

110

120

130

MAEL domain (residues 84–333)
α3

α2
150

140

160

170

180

190

β4
200

η2

210

α4
220

IYSTMIDPGQIIFGQGSDALLHSSTTHDLPLPPNALGEKNMTKLYRNIVDYLSKCQ.GKGKTLVVFTPAENITMVKSCFRYLESYHETIIPSCVPVGYMFDVKLG.AEEFGLEMPGTDDAGPNYIQILANIIDYLKQKDRTVQVLPPMFTLPEKVDAVQNFISQMCSFHDFIDPGELPRGFRYHCQSGSASTHQIPISGFELANSDYHNMFRKLCSFVCP...TPCPVVPVYTKANDIYRVDWCLQWLADFHSFIHPGEIPRGFRFHCQAASDSSHKIPISNFEFG.HDQATVLQNLYKFIHP...NPGNWPPIYCKSDDRARVNWCLKRMEDFHSFINPGEIPRGFRFHCQAASDSSHKIPISNFERG.HNQATVLQNLYRFIHP...NPGNWPPIYCKSDDRTRVNWCLKHMA

α5

β5

230

Dm
Bm
Xt
Mm
Hs

60

NGHGQVDKAQREATES....LMDMKRTIERLVLNAKMSHDLENAKFVFVAFNYFTKALT..TDVYVPAEFAACEYSLKEGIRSSTGIPIKVIEQQQREMKNAEDNEKKDIQNIVKLKVF.DQSTKTEDFYVIDVNSYCKAN....GDYLIGEFTVTQFSLQDGVKNTKSSDFSSVGFFQLNDQRNCVTQRTVPAMNIDISFNKNKAPDQHVCYFINIFSHGDMPSLCEQRYVPCEIACVRYSLREGILGLVSTPLRRPGMLVPK.......PSISPPDMSNLSIKSDQALLGGIFYFLNIFSHGELPPHCEQRFLPCEIGCVKYSLQEGIMA
PVFTPLRRPGMLVPK.......QNVSPPDMSALSLKGDQALLGGIFYFLNIFSHGELPPHCEQRFLPCEIGCVKYSLQEGIMA

β3
Dm
Bm
Xt
Mm
Hs

50

MAPKKHSG..FMMFVNEWRNRNAEG..RRMTLAQAVSHCGTIWEKMNTQQRGPYNSGGKDANVAQRAKR...........ESSMPKKKTPQNPFFFFMMDYRKEQAEIGIKYANTKELAEAAGPVWQNLRPTLKAKYEEKAKNEKEKNKQTG..........TKFTMPNKKAARNAYFFFALDMIPELRRRGLQVSGVREAIPLCSEDWALLSADQKEAYAEKARLLKNDPDSTSTYNDGVFGQRKRNAMPNRRASRNAYYFFVQEKIPELRRRGLPVARVADAIPYCSADWALLREDEKEKYSEMAREWRAAQGKDS.......GPSEKQKMPNRKASRNAYYFFVQEKIPELRRRGLPVARVADAIPYCSSDWALLREEEKEKYAEMAREWRAAQGKDP.......GPSEKQK

α6

240

α7

250

260

270

α8
280

290

η3

α9

300

310

CDDDFRDGGEKIQVFDIQYLLFILKKEVMNVADLNDEKINKFATDAFFKKDFFEFTAGIACQYHEDNDRTKYCTQSMVTRWAYNCATEDDS..LFRIYKLDTFFFTLINAISSHHDEG..FPKESLALTQLTKDLFDYTPGIACERHESLDRSNVCTTSRVKRWVFNKAGMEN...HFRVQEVETLIIKFYQDKLQEE......PSRPTVSRLLDVVQWDYSSNTRCKWHEDND.MWCCALASCKKIAYRASEIRQ...DLELLTVEDLVVGIYQQKFLKE......PSKTWVRSLLDVAMWDYSSNTRCKWHEEND.ILFCALAVCKKIAY
KASEIRQ...DLQLLTVEDLVVGIYQQKFLKE......PSKTWIRSLLDVAMWDYSSNTRCKWHEEND.ILFCALAVCKKIAY

η4
320

330

340

350

360

370

S

380

390

Dm
Bm
Xt
Mm
Hs

TFTDFMCGDLAITVQPGKHIPAQTKPN.YLIISSYASSLDHESSFDSFYSLPGSGVKKESQPEACLSSSRLSVASSSYKPIDH
TILDRCCPLLGIPLQPGKHLPFDYDINVSTGQSAADQRSRAPCISKALASVYGVTLTPA.HLPNPERSRNQNSVNPKVVVLDAKRFQRVCSSDP...KYSTDKSERSSFEPRGVKPYQGPSGG...
CISNSLATLFGIQLTGA.HVPLQDYEAS.NSVTPKMVVLDAGRYQKLRVESPGFCHFNSYNQEQRSNTSTGYYPSGVKISGPHSCISNSLATLFGIQLTEA.HVPLQDYEAS.NSVTPKMVVLDAGRYQKLRVGSSGFSHFNSSNEEQRSNTPIGDYPSRAKISGQNS

Dm
Bm
Xt
Mm
Hs

TSFAANLNEVSEFPSLGMRNSSKHHGIAASAQREWNARNLPTHSRLIRKVSDNDFSVNGADGKLKK

400

410

420

430

440

450

...GRGILRLLETLAASQNSSGSVRGRGITRLLESISNSSNNIHRFSSCETSLSPYTPQKDGYKPFSSFSSVRGRGITRLLESISNSSSNIHKFSNCDTSLSPYMSQKDGYKSFSSLSECHC motif
Residues corresponding to the DEDDh motif
Residues located at the central groove
Residues involved in the ssRNase activity
Residues not involved in the ssRNase activity
Cysteine residue substituted with serine for crystallization

Figure S1. Multiple amino acid sequence alignment of Mael orthologs from higher eukaryotes, Related to Figure 1
Multiple sequence alignment of Mael orthologs from Drosophila melanogaster (Dm), Bombyx mori (Bm), Xenopus tropicalis (Xt), Mus musculus (Mm) and Homosapiens (Hs). The secondary structure of DmMAEL is indicated above the sequences. The ECHC-motif residues are indicated by yellow triangles. Residues equivalent
to the DEDDh motif are indicated by pink triangles. Residues at the central groove are indicated by gray triangles. Cys228, which was substituted with serine forcrystallization, is indicated by a black triangle. Residues involved in the ssRNase activity are indicated by red triangles, whereas residues not involved in the ssRNase
activity are indicated by cyan triangles.

α1
70

Dm
Bm
Ag
Cq
Aa
Tc
Am
Bt
Hs
Bi
Dp

80

η1

90

100

β1

β2

110

120

130

140

KRESSNGHGQVDKAQREATESLMDMKRTIERLVLNAKMSHDLENAKFVFVAFNYFTKAL...TTDVYVPAEFAACEYSLKFTSTGIPIKVIEQQQREMKNAEDNEKKDIQNIVKLKVFDQSTKTEDFYVIDVNSYCK.....ANGDYLIGEFTVTQFSLQITNIGIPISEITQEKRDRESKAERLKKLVSTLVMNAASKNVLEKQEFYFISMAYFCRT....NTGVHLPAELAVVRYSLEYTAQGKPFSQVQAEAAKRRQLEEQIQKRIVEMIQTASANNALGELEVYFVSCNYFCVS....LSGEYVPAELAIIKYSLNYTAQGIAFSQVEMEKQQLLKKHETIRKTISEMIQTAVLNNALEKLEVFFMSCNYFCKT....STEAFVPAEIALIKYNLE
YTTDGIDIEVVERKERDEARKKQEMKDDITRTLKAAYFATDLDEKIFLVIHINHLAYYP...TEDKYFICEIAIAAVSLKKTTIGEDLTEVELNLKREQEFQQKMLQYIDSVVSMGLLHNNLQKLKFIFIHVNWFYKREIGINKCEFCPAEFAVAQFSLERTTIGENVAQLELEQKREEEFQQNMLQYIESIVCMGVQHNNLQNLKFIFIHVNWFYRRDIGINKYDFCPAEFAVAEFSIQKTTHGECIQLLEQKEIEEQKFIEDMYEYLESTITRAVNHNVLPQLKFCFVHVNWFFTKLVDN.VVEYFPAEYAVGVFSLE
RTTIGESVAELELEQKRQEEFQQNMLQYIESIVGVGVRHDNLKNLKFIFIHVNWFYRRDIGINKYDFCPAEFAIAEFSIL
FTSTGIPISVIEQNEREAKEAEEQEIQDIKNLVKLRGFNNTAKTLDIYVMDVNCYCK.....TGSDYIIGESTLLRFNVE

MAEL domain (residues 84–333)
β3

α2
150

Dm
Bm
Ag
Cq
Aa
Tc
Am
Bt
Hs
Bi
Dp

α4

Dm
Bm
Ag
Cq
Aa
Tc
Am
Bt
Hs
Bi
Dp

α3

170

180

β4

190

200

η2

210

EGIRSIYSTMIDPGQIIFGQGSDALLHSSTTHDLPLPPNAL.GEKNMTKLYRNIVDYLSKCQGKGKTLVVFTPAENITMVDGVKNSYHETIIPSCVPVGYMFDVKLGAEEFGLEMPGTDDA.GPNYIQILANIIDYLKQKDRTVQVLPPMFTLPEKVDAVGGVKDKLHMFINPGRLPIGMAYDAQRHAEEDHQLPLPPNAM.GVSDYGDVAMRLFSFLLQNDD...MPLLFTDETDVPRV
DGVMDSLNVLINPTDLPLGMALDAKTHSSSTHQLPVPPDAL.GEANYEKILRQILKFFKNTSGSKVVPPIFTWNKDIPMVLGVLDKLHELINPVRLPLGLAHEALTYSEQTHELPTPPNAM.GETDFYTVLQKILSFTDYNSKPHKKLAIMTDAKEVPVINGVEDVFHRIVKPGKLPLGYYGGALTHSKETHQMLELVQDEPYENNTREVFNEMTSFLKLWRGKGSDSIVYADEKTHEMINGVENIYHEVLK.MKIPLGWKRDAIETSQQT.HQIPIELED.GQSDFSYMFNELTKFLESNKTGNKFPPLFTAKDLSPVVNGIQNIYHEILK.VKIPLGWKRDALETSQET.HQIPIELDD.GECDFALMYNKFSKFMKSNKTGRRYPPLFTMKNLCPVVNGIEDIHHTIVG.VPIPLGYRREALETSQST.HNIPIECPG.EESDFAIMYEKLTNFLKPRKIVDKYPPLYTTKSLKGAVNGIRNVYHEILK.VKIPLGWKRDALETSQET.HQIPIELDD.GEYDFALMYDKFSKFMERNKTGRRYPPLFTLKNAYPIVEGIRDTYHEIINPGVIPVGYAYDMKLSCKEFGLEMPDECSK.KSNYMQILANIIDYLKQQDRTSKVLPPIFTMPDKVAPV

α5

220

Dm
Bm
Ag
Cq
Aa
Tc
Am
Bt
Hs
Bi
Dp

160

230

β5
240

α6
250

α7
260

270

α8
280

290

KSCFRYLECDDDFRDGGEKIQVFDIQYLLFILKKEVMNVADLN...DEKINKFATDAFFKKDFFEFTAGIACQYHEDND.
QNFISQMCNCATEDDS..LFRIYKLDTFFFTLINAISSHHDEGFPK.....ESLALTQLTKDLFDYTPGIACERHESLD.
ESMLEHILSD.HLSEI..ELRICPLAELFFRLKQNVELYMMD...QTTFPSVYIAQQIITKDVYDYTKGISCDYHEEKD.
DSILRGILEATDLDYV..KFSILPLIDFFYNLKLATEDYGLD...IKTFPSIHLAKALLEKDVYAYTAGIACDVHEQLN.
ESLLSQLNDDVKLEY...QFLVIPLGEFFFHLKRATEKYGLD...ICTFPTKTVADILLKKDAYEYTSGIACDFHEKLG.
TKVIDNFCQEFNYPD...EIKVYNFQYLFFALRNSVAARTVWP.......TETYSSTELEKDLYSYTPDISCEFHEMSD.
ESLLIKMIDASNGSID..DFIIYSIEALFGALRNAAVQKVDD...RSIP..LIVAENEFSKDFLCNTRGLECDFHKILD.
ESLLQRLNEASGESVD..DYLIYSIEALFAELRNAAVRSVDN...RSIP..LVLAEMEFAKDFLSSTRGFECEFHKIVD.
QSLLATLCKAANQDEE..QFLVYEIESLFKHLADEAYKKRED...REIKYTKAYAEYTFTRFTHIYKRGNECIFHKYTDG
ESLLQRLNEASGQSVD..DYLIYSIEALFGELRNAAVRKIDK...RGIP..LVLAETEFAKDYLSTMRGFECEFHKIVD.
QNFIHQLCRRVAEDEN..QFRIYKLDTLFFTLINTLKTRADEGFPK.....ESLALLQLKKDPFKYSPGIGCEHHEEND.

η3

α9

η4

300

310

320

330

340

RTKYCTQSMVTRWAYTFTDFMCGDLAITVQPGKHIPAQTKPNYLI...
RSNVCTTSRVKRWVFTILDRCCPLLGIPLQPGKHLPFDYDINVST...
NVLYCPLSRCIRWAYIISDNCCQDMGIEPIPGKHVPLNANTNPTA...
NQVACALSRVVRWAYVISDSCCLDVGIEMEKGRHLPHNMTTLSDI...
NQRFCALSKVVRWSYIISDNCCLDLSIDLIAGRHLPSNADTTLCS...
ISVYCSKSIVTRYCYTLCDHCCTDLNIQLVAGFHVPKNSRIAVDS...
ISQYCSKSIVKRWAFTICDYCCEYLNIKLIEGVHRPKETPFLQSS...
VPQYCSKSIVTRWGMTICDYCCEYMNIEMIEGVHCPYKNSLEFNK...
GSEYCSKSVLHQWTWNMCEEFCNPLQVEMKSGVHYPEEADCSYKG...
VAQYCSRSIITRWGMTICDYCCEYLNVEMIEGVHRPYKNSFDINR...
KSVECTLSRVKRWSYTVLDACCPVAGVTARPACHVPPDYDLESIQ...

ECHC motif
Residues corresponding to the DEDDh motif
Residues located at the central groove
Residues involved in the ssRNase activity
Residues not involved in the ssRNase activity
Cysteine residue substituted with serine for crystallization

Figure S2. Multiple amino acid sequence alignment of the MAEL domains from insect species, Related to Figure 1
Multiple sequence alignment of the MAEL domains from Drosophila melanogaster (Dm), Bombyx mori (Bm), Anopheles gambiae (Ag), Culex quinquefasciatus (Cq),
Aedes aegypti (Aa), Tribolium castaneum (Tc), Apis mellifera (Am), Bombus terrestris (Bt), Harpegnathos saltator (Hs), Bombus impatiens (Bi) and Danaus plexippus(Dp). The color code of the triangles is the same as in Figure S1.

A

His291
His509His291
His509Cys288Cys506

Cys288Cys506

Cys300Cys529Cys300Cys529Glu131Glu399Glu131Glu399B
Thr171
Ser430Thr171
Ser430
Asn116
Glu391

Asn116
Glu391
Met218
Asp466

Tyr299His528

Mn2+

Asn215

Met218
Asp466

Tyr299His528

Mn2+Mn2+

Asn215
Ala114
Asp389

Met304
Asp533

Ala114
Asp389Arg307

Met304
Asp533Arg307

Thr211

Thr211
Pro212
Gln462

Arg492
Asp278Trp308

Pro212
Gln462

Arg492
Asp278Trp308Figure S3. Structural comparison of DmMAEL with LASV NP, Related to Figure 1(A) Stereo view of the superimposition of the ECHC motifs of DmMAEL (light blue) and LASV NP (gray). The bound zinc ions are shown as spheres.
(B) Stereo view of the superimposition of the central groove of DmMAEL (light blue) and the catalytic groove of LASV NP (gray). The manganese ions bound to LASVNP are shown as gray spheres.

DmMAEL

Rescue plasmid

EGFP

siMaelDmMAEL WT

FL-DmMael WT

Rescue plasmid

EGFP

siEGFP siMael

EGFP

A

Myc-EGFP
DmMAELFL-DmMaelendo FL-DmMael

Anti-Myc

Anti-Mael
DmMAEL

Anti-Piwi

Anti-TubsiMael

B

C300A

H291A

C288A

E131A

ECHC

WT

Rescue plasmid

EGFP

FL-DmMael

FL-DmMael
Anti-Maelendo FL-DmMael
Anti-TubC

siMaelRescue plasmid

EGFP
WT
ECHC
K140A
K199A
Q289A
D293A
D295A
D314A
K328A

DmMAELendo FL-DmMael
Anti-Mael
DmMAEL
Anti-Piwi

Figure S4. Expression levels of Mael in Drosophila OSCs, Related to Figure 4
The expression levels of wild-type FL-DmMael and DmMAEL (A), the ECHC-motif mutants of FL-DmMael (B), the ECHC-motif quadruple mutant and the ssRNasedeficient mutants of DmMAEL (C) were examined by western blotting using an anti-Mael or anti-Myc antibody. Tubulin or Piwi was used as a loading control. WT, wild
type.

Table S1. Data Collection and Refinement Statistics, Related to Figure 1
DmMAEL(Crystal I)

DmMAEL(Crystal II)

Beamline
Wavelength (Å)

SPring-8 BL32XU
1.282

SPring-8 BL32XU
1.000

Space group

P43212

P43212

71.9, 71.9, 86.8

71.9, 71.9, 88.4

Data collection

Cell dimensions
a, b, c (Å)α, β, γ (°)

90, 90, 90

90, 90, 90

50.0–2.40 (2.55–2.40)
0.157 (0.897)

50.0–1.60 (1.70–1.60)
0.088 (1.17)

I/σI

8.13 (2.01)

18.7 (2.40)

Completeness (%)
Redundancy
CC(1/2)92.8 (95.3)
4.08 (3.86)
0.992 (0.715)

99.8 (99.6)
14.3 (14.3)
0.999 (0.797)

Resolution (Å)
Rsym

Refinement
Resolution (Å)

33.4–1.60

No. reflections

31,075

Rwork / Rfree

0.194 / 0.216

No. atoms
Protein

1,902

Ligand8

Zinc ion

1

Solvent

82
2

B-factors (Å )Protein

31.0

Ligand43.8

Zinc ion

17.9

Solvent

34.4

R.m.s. deviations
Bond lengths (Å)

0.011

Bond angles (º)

1.289

Ramachandran plot
Favored (%)

98.3

Allowed (%)

1.7

Outlier (%)

0.0*Highest resolution shell is shown in parentheses.

Table S2. Oligonucleotide Sequences, Related to Figures 2 and 4
Oligonucleotides for nuclease activity measurement
40-nt sense RNA

GGUCUGAUUUCGAUCUGGUUCCCUGGAACAAAAGUGGCAG

40-nt antisense RNA

CUGCCACUUUUGUUCCAGGGAACCAGAUCGAAAUCAGACC

40-nt sense DNA

GGTCTGATTTCGATCTGGTTCCCTGGAACAAAAGTGGCAG

40-nt antisense DNA

CTGCCACTTTTGTTCCAGGGAACCAGATCGAAATCAGACC

15-nt poly(A)AAAAAAAAAAAAAAA

15-nt poly(A) with 1G

AAAAAAAGAAAAAAA

15-nt poly(A) with 3G

AAAAAAGGGAAAAAA

siRNAs for rescue experimentsiMael sense

CGCCAAGAUGUCCCAUGAUTT

siMael antisense

AUCAUGGGACAUCUUGGCGTT

siEGFP sense

GGCAAGCUGACCCUGAAGUTTsiEGFP antisense

ACUUCAGGGUCAGCUUGCCTT

Primers for qRT-PCR analysis
mdg1_forward

AACAGAAACGCCAGCAACAGC

mdg1_reverse

CGTTCCCATGTCCGTTGTGAT

297_forward

CTGGCAAAGGGATTTCATCA

297_reverse

TGCATTCCTAAGGCCAAATG

blood_forward

TATCGCATGGCAGATAGCCAAA

blood_reverse

CGTGGAATTCGGAAGTGGTTTC

Tabor_forward

ACGTTGTTCACGACATTAGCCG

Tabor_reverse

GGGTTGGTTCGGATCTGACG

gypsy_forward

ACTAGACTGCACGTACTCGGACA

gypsy_reverse

GTTAGCTTCTTTTCAACTTCATCGTZAM_forward

ACTTGACCTGGATACACTCACAAC

ZAM_reverse

GAGTATTACGGCGACTAGGGATAC

HeT-A_forward

CGCGCGGAACCCATCTTCAGA

HeT-A_reverse

CGCCGCAGTCGTTTGGTGAGT

rp49_forward

CCGCTTCAAGGGACAGTATCTG

rp49_reverse

ATCTCGCCGCAGTAAACGC

Primers for cloning
Bm-forward

TTTTTGGTACCATGCCAAAAAAGAAGACCCCACA

Bm-reverse

TTTTTGCGGCCGCAGGGTGCTCTGGACCTTTG

Mm-forward

TTTTTGGATCCCCATGCCCAACCGCAGGGCC

Mm-reverse

TTTTTGTCGACTTAAGAAAAGGAGGAGAAAGGSUPPLEMENTAL EXPERIMENTAL PROCEDURES
Protein preparation
The gene encoding D. melanogaster Mael (residues 1–459, referred to as FL-DmMael) was amplified by
PCR using pBlueScript-FL-DmMael (Sato et al., 2011) as the template. The PCR product was cloned
between the NdeI and XhoI sites of a modified pET28a vector (Novagen), in which the thrombin proteaserecognition sequence was replaced with the Turbo3C Protease recognition site. FL-DmMael was
expressed in E. coli Rosetta2 (DE3) cells (Novagen), and was purified by chromatography on Ni-NTA
Superflow (QIAGEN), Resource Q (GE Healthcare) and Superdex 200 10/300 (GE Healthcare) columns.
We only obtained a small amount of purified FL-DmMael (0.03 mg from 1 liter E. coli culture), due to its low
expression levels. To identify a well-expressed, stable region suitable for structural and biochemical
studies, we performed limited proteolysis experiments. Purified FL-DmMael was digested with trypsin(Sigma) (20:1, w/w) at 26°C for 20 min, and the products were resolved by 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), followed by N-terminal amino acid and mass
spectrometric analyses. The limited proteolysis experiments revealed that the MAEL domain of D.
melanogaster Mael (residues 84–333, referred to as DmMAEL) is a stable 28 kDa fragment.
The gene encoding DmMAEL was cloned between the NdeI and XhoI sites of the modified
pET28a vector, and DmMAEL was overexpressed as an N-terminal His-tagged protein in E. coli Rosetta2(DE3) cells. The E. coli cells harboring the expression plasmid were cultured at 37°C in LB medium,-1
containing 50 mg l kanamycin. Protein expression was induced by the addition of 0.1 mM isopropyl-β-Dthiogalactopyranoside, when the culture reached an OD600 of 0.8. The E. coli cells were further cultured at
15°C for 18 h, and were harvested by centrifugation. The E. coli cells were resuspended in buffer (50 mM
Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole, 3 mM 2-mercaptoethanol, 5% glycerol), lysed bysonication, and centrifuged. The supernatant was incubated with 3 ml Ni-NTA Superflow resin, and the
mixture was then loaded into an Econo-Column (Bio-Rad). The protein was eluted with buffer (20 mM TrisHCl, pH 8.0, 300 mM NaCl, 300 mM imidazole, 3 mM 2-mercaptoethanol, 5% glycerol). The eluted protein
was treated with Turbo3C Protease (Nacalai Tesque) at 4°C for 16 h to cleave the N-terminal His tag, andthen was passed through the Ni-NTA column again. The protein was loaded onto a Resource Q column,
equilibrated with buffer (20 mM Tris-HCl, pH 8.0, 30 mM NaCl, 1 mM DTT), and was eluted using a linear
gradient of 30–600 mM NaCl. The purified protein was dialyzed against buffer (10 mM Tris-HCl, pH 8.0,
150 mM NaCl, 1 mM DTT), and was concentrated to 5 mg ml -1 using an Amicon Ultra 10K filter (Millipore).
DmMAEL generated crystals under several conditions, but the crystals diffracted poorly. We thus
prepared the five less-conserved cysteine mutants (C135V, C200S, C222S, C228S and C317A), using a
PCR-based method. The sequences were verified by DNA sequencing. All of the cysteine mutants wereexpressed in E. coli, and were purified by a protocol similar to that for wild-type DmMAEL.
Crystallization
Crystallization was performed at 20°C, using the sitting-drop vapor diffusion method. Of the five cysteine
mutants, only the DmMAEL C228S mutant generated well-diffracting crystals. We obtained crystals of the-1
DmMAEL C228S mutant (Crystal I), by mixing 0.1 μl of protein solution (5 mg ml DmMAEL, 10 mM TrisHCl, pH 8.0, 150 mM NaCl, 1 mM DTT) and 0.1 μl of reservoir solution (200 mM ammonium acetate, 20%
PEG3350). Furthermore, we obtained better-diffracting crystals (Crystal II) by the streak-seeding technique,using a slightly different reservoir solution (200 mM ammonium acetate, 19% PEG3350).
Data collection, structure determination and refinement
Crystals were cryoprotected by the reservoir solution supplemented with 25% ethylene glycol. X-ray
diffraction data sets were collected at 100 K, on the beamline BL32XU at SPring-8 (Hyogo, Japan).
Diffraction data were processed using XDS (Kabsch, 2010). We performed zinc K-edge X-ray absorption
fine structure experiments, and found that the crystal contains endogenous zinc atoms. Diffraction data
sets for Crystal I (2.4 Å resolution) and Crystal II (1.6 Å resolution) were collected at wavelengths of 1.282
Å and 1.000 Å, respectively. The crystal structure of DmMAEL was determined by the SAD method, using
the 2.4 Å resolution data set from Crystal I. One zinc site was located by the program SHELXD (Sheldrick,2008), and the experimental phases were calculated with the program SHARP (Bricogne et al., 2003). The
atomic model was automatically built with the program RESOLVE (Terwilliger, 2003). The resultant modelwas manually built with the program COOT (Emsley et al., 2010), and refined with the program PHENIX(Adams et al., 2002). The final model was refined at 1.6 Å resolution, with Rwork/Rfree of 19.4%/21.6%,
using the data set from Crystal II. A summary of the crystallographic data collection and refinement
statistics is provided in Table S1. Structural figures were prepared with the program CueMol(http://www.cuemol.org).
Nuclease activity measurement
The gene encoding B. mori Mael (residues 1–354) was amplified by RT-PCR from total RNA from B. moriovary-derived BmN4 cells, and cloned into the pGEX-5X-1 vector (GE Healthcare). Using this vector as the
template, the expression plasmid encoding the MAEL domain of B. mori Mael (residues 92–335, referred
to as BmMAEL) was prepared in a similar manner as described for DmMAEL. The wild type and mutantsof DmMAEL and BmMAEL were expressed in E. coli, and the proteins were purified by chromatography on
Ni-NTA Superflow and Resource Q columns, using a protocol similar to that for DmMAEL. To avoid thepotential cleavage of nucleic acid substrates by contaminants from E. coli, the proteins were further
purified by chromatography on Resource PHE (GE Healthcare) and Superdex 200 10/300 (GE Healthcare)columns. The gene encoding M. musculus Mael (residues 1–434) was amplified by RT-PCR from total
RNA from C57BL/6J line mouse testis (Japan SLC, Inc), and cloned into the pGEX-5X-1 vector. Using thisvector as the template, the expression plasmid encoding the MAEL domain of M. musculus Mael (residues
83–327, referred to as MmMAEL) was amplified by PCR, and the PCR product was cloned into a modified
pFastBac HTa vector (Invitrogen), in which the SUMOstar tag (LifeSensors) was inserted between thehistidine tag and TEV protease cleavage site. MmMAEL was expressed in Sf9 insect cells. Baculovirusinfected Sf9 cells were cultured in Sf900II (Invitrogen) at 27°C, and were harvested 60 h after infection by
centrifugation. The insect cells were resuspended in buffer (20 mM phosphate buffer, pH 7.3, 300 mM
NaCl, 20 mM imidazole, 3 mM 2-mercaptoethanol, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride), lysedby sonication, and ultracentrifuged. The supernatant was incubated with Ni-NTA Superflow resin, and
MmMAEL was eluted with buffer (20 mM phosphate buffer, pH 7.3, 300 mM NaCl, 300 mM imidazole, 3mM 2-mercaptoethanol, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride). The eluted protein was treatedwith TEV protease at 4°C for 16 h to cleave the N-terminal His-SUMOstar tag, and was passed throughthe Ni-NTA column again. The protein was further purified by chromatography on Resource Q, Resource
PHE and Superdex 200 10/300 columns, using a protocol similar to those for DmMAEL and BmMAEL. The
purified proteins were analyzed by 15% SDS-PAGE, and stained with SimplyBlue Safestain (Invitrogen).
The nucleic acid substrates used for the activity measurements are listed in Table S2. The
ssRNAs and ssDNAs were purchased from GeneDesign and Eurofins Genomics, respectively. The
32
ssRNAs and ssDNAs were 5′-radiolabeled with P-γ-ATP and T4 polynucleotide kinase (New England
Biolabs), and were purified by 15% denaturing (6 M urea) PAGE. To prepare the dsRNA and dsDNA
substrates, the radiolabeled antisense strands were mixed with a 2-fold excess of the non-labeled,complementary sense strands in buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA), heated at
95°C for 15 min, and then gradually cooled to 30°C. To prepare the circular ssRNA substrate, theradiolabeled ssRNA was incubated at 37°C for 15 min with ATP and T4 RNA ligase 1 (New England
Biolabs) in buffer (50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 1 mM DTT), and then purified by 10%
denaturing (6 M urea) PAGE. Prior to the cleavage reactions, all of the radiolabeled single-stranded
substrates were unfolded by an incubation at 95°C for 2 min, followed by immediate cool down at 4°C.
4The purified DmMAEL (2.2 μM) was incubated with substrates (10 c.p.m.) at 26°C for 3 h, in 20
μl of reaction buffer (25 mM HEPES-KOH, pH 7.4, 5 mM DTT). The reaction mixture was mixed with anequal volume of loading buffer (98% w/v deionized formamide, 10 mM EDTA, pH 8.0, 0.025% w/v xylene
cyanol, 0.025% w/v bromophenol blue), heated at 95°C for 2 min, and then analyzed by 15% denaturing (6M urea) PAGE. The radioactive signals were detected using a Typhoon FLA 9500 image analyzer (GEHealthcare). The effects of MgCl 2 and CaCl2 on the nuclease activity were examined using reaction buffers(25 mM HEPES-KOH, pH 7.4, 5 mM DTT, 3 mM MgCl 2 or CaCl2). The effects of NaCl were examinedusing reaction buffers (25 mM HEPES-KOH, pH 7.4, 5 mM DTT, 0–100 mM NaCl). Exonuclease T (7.5
units) (New England Biolabs) was incubated with the circular ssRNA in reaction buffer (50 mM potassium
acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT). The effects of the substrate
structure on the nuclease activity were examined using 15-nt poly(A) containing 0, 1 or 3 guanine residues.
To compare the enzymatic properties of DmMAEL and RNase T1, the ssRNase activities of DmMAEL(0.14–2.2 μM) and RNase T1 (Ambion) (0.5–10 units) were measured using the 40AS ssRNA as thesubstrate. The ssRNase activities of DmMAEL (1 μM) and BmMAEL (1 μM) were measured at 26°C for 3 h,and that of MmMAEL (1 μM) was measured at 37°C for 3 h, using the 40AS ssRNA as the substrate. To
identify the residues involved in ssRNA cleavage, the ssRNase activities of the wild type and mutants of
DmMAEL (1 μM) were measured at 26°C for 1 h, using the 40AS ssRNA as the substrate.
Rescue experiments in OSCs
The gene encoding FL-DmMael was amplified by RT-PCR from total RNA from Drosophila ovary, and wascloned between the KpnI and XhoI sites of the pAcM vector (Saito et al., 2010). The rescue plasmids
encoding the RNAi-resistant, wild type and mutants of FL-DmMael were prepared by a PCR-based
method, using pAcM-FL-DmMael as the template. The rescue plasmids encoding the wild type andmutants of DmMAEL were prepared by a PCR-based method, using pAcM-FL-DmMael as the template.
pAcM-EGFP was used as a negative control (Saito et al., 2010).
Transfection was performed essentially as described previously (Saito et al., 2010). Briefly,
6
trypsinized OSCs (3×10 cells) were transfected with the siRNA duplex (300 pmol) and rescue plasmid (5μg) using Nucleofector (Lonza), and then transferred to fresh OSC medium. After an incubation at 26°C for
2 days, total RNA was purified using the ISOGEN reagent (Nippon Gene) or the RNAzol RT reagent(Cosmo Bio). cDNAs were synthesized from 1 μg total RNA, using a Transcriptor First Strand cDNA
Synthesis Kit (Roche Diagnostics) and an oligo(dT) primer. qRT-PCR was performed with SYBR Premix
Ex Taq (Takara) or THUNDERBIRD SYBR qPCR Mix (TOYOBO), using a LightCycler Real-Time PCRsystem (Roche Diagnostics) or a StepOnePlus system (Applied Biosystems). The amplification efficiencyof the qRT-PCR was calculated, based on the slope of the standard curve. After confirmation of theamplification efficiency values (between 95% and 105%), the relative steady-state mRNA levels were
determined from the threshold cycle for the amplification, using ribosomal protein 49 (rp49) as an internal
control. The expression levels of DmMael, Piwi and tubulin were analyzed by western blotting, using theculture supernatants of anti-Mael hybridoma cells (Sato et al., 2011), anti-Piwi hybridoma cells (Saito et al.,2006), and an anti-tubulin antibody (Developmental Studies Hybridoma Bank, E7), respectively. The
siRNA duplexes and the qRT-PCR primers are listed in Table S2.

SUPPLEMENTAL REFERENCES
Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J.,
Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX : building new software for automatedcrystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954.
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