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, 1–10
April 21, 2015 ª2015 The Authors
http://dx.doi.org/10.1016/j.celrep.2015.03.030

Accession Numbers
4YBG
LC032360

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

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,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
Cell Reports 11, 1–10, April 21, 2015 ª2015 The Authors 1

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

A

HMG
1

C

MAEL
71 84

333

His291459

DmMAEL

Glu131B
α2Cys300

ECHC motif

Zn2+
Cys288α4α5η2N (84)
β5

α3

D

α8

β1 β2

dsRNA

η3
α9

β4
α7

β3

Zn2+

Zn2+

Mn2+

C (332)α1α6η1LASV NP

η4
DmMAELE

F
Cys288Cys506

Thr171

His291
His509

Asn116

Ser430Glu391

Tyr299

Met218His528

Asp466

Zn2+
Zn2+

Cys300Cys529

Mn2+

Asn215
Arg307
Ala114

DmMAELLASV NP

Glu131Glu399Thr211

Asp389
Met304

Asp533
Gln462

Pro212
Trp308LASV NPECHC motif

ECHC motif
dsRNA

180˚

dsRNA

180˚

Catalytic groove
Central groove

Arg492

Asp278DmMAELG

Mn2+

LASV NP

DmMAEL(legend on next page)2 Cell Reports 11, 1–10, April 21, 2015 ª2015 The AuthorsPlease cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

et al., 1997, 2001; Pek et al., 2009, 2012; Sato et al., 2011). Maelis composed of an N-terminal HMG domain and a central MAEL
domain, which was predicted to adopt an RNase H-like fold by abioinformatics 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 substi-

tution 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, theFigure 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, 1–10, April 21, 2015 ª2015 The Authors 3

B

Absorbance at 280 nm

Fraction
1 2345678

C

D

DmMAEL (µM)

DmMAEL

Metal ion

Time (min)

(nt)
50
40
30
10

15

20

180
15
30
60
120
180

A

0.14
0.28
0.55
1.1
2.2

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

(nt)

(nt)

50
40

50
40

30

20

Mg2+

Ca2+

-

-

+

+

30

24

Elution volume (ml)-DmMAEL +

20
20

(kDa)
250
150
100
75
50
37

40AS ssRNA

40AS ssRNA

F

Linear Circular
DmMAELE

1540AS ssRNA

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

10

Fraction 1

2

3

4

5

6

7

DmMAEL

8

- +

- +

- +

- +

- +

(nt)

(nt)

(nt)

40

60
50

50

40

40

30

30

30

20

ExoT

25
20

G

15-nt poly(A)
0G

1G

3G

DmMAEL - + - + - +
(nt)

40
30
20

20

20

10

*
40AS ssRNA

40AS ssRNAJ
No enzyme

1

(nt)

NaCl

DmMAEL
NaCl

40

6

30

5
4

20

3

NaCl

(mM) 0 25 50 100 0 25 50 100 0 25 50 100
(nt)

2

50

RNase T1

50
40

3

4

30

-

(kDa)
250
150
100
75

(nt)

50

30

DmMAEL
BmMAEL
MmMAEL

RNase T1I

DmMAEL
BmMAEL
MmMAELDmMAEL

5′-CUGCCACUUUUGUUCCAGGGAACCAGAUCGAAAUCAGACC-3′

H

40

37
20

20

25
20

5

2
1
40AS ssRNA

10
15

6

10

40AS ssRNA

40AS ssRNA(legend on next page)

4 Cell Reports 11, 1–10, April 21, 2015 ª2015 The AuthorsPlease cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

central 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 of
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
the sequence conservation among Mael orthologs. However,multiple sequence alignments indicated that only the ECHC
motif is solvent accessible and strictly conserved across theMael orthologs (Figures S1 and S2). Thus, based on the crystal
structure of DmMAEL, we prepared 12 DmMAEL mutants, inwhich 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 not shown), confirming their structural integrity. We thenexamined the ssRNase activities of the purified mutants, using
40AS ssRNA as the substrate (Figure 3B). The K109A, K188A,
N192A, and E292A mutants showed ssRNase activities comparable to that of the wild-type DmMAEL, and the K277A mutant
showed moderately reduced ssRNase activity (Figure 3B).

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, 1–10, April 21, 2015 ª2015 The Authors 5

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

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˚
15

Asp314
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]).

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 ssRNA in 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 theseresidues are involved in ssRNA binding, but not in catalysis.
The positively charged residues, Lys140, Lys199, and Lys328,
would interact with the negatively charged phosphate backboneof the ssRNA substrates. These residues are located on the
opposite side of the central groove, which is equivalent to thecatalytic groove of LASV NP (Figures 3C and 3D). To examine
whether the central groove is involved in the ssRNase activity,
we tried to prepare four additional DmMAEL mutants (N116A,
6 Cell Reports 11, 1–10, April 21, 2015 ª2015 The Authors

M218A, Y299A, and M304A), in which the residues corresponding to the DEDDh motif were individually 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 thessRNase activity (Figure 3B), indicating that the C228S mutation
does not have considerable impact on the structure and functionof 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 througha metal-ion-independent mechanism, in which a conserved histidine serves as a 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

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

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

2

2
1

1

1

0

0

100

0

FL-DmMael

DmMAEL

EGFP
WT
ECHC
D295A
D314A

0

EGFP
WT
ECHC
K140A
K199A
Q289A
D293A
K328A

1

Rescueplasmid

B

2

3

EGFP
FL-DmMael
DmMAEL

Relative mdg1 expression

2

EGFP
WT
ECHC
E131A
C288A
H291A
C300A

A

(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

Relative expression

To examine whether the ssRNase
activity of DmMAEL is involved in Piwi/
80
Mael-mediated TE silencing, we next
overexpressed the seven ssRNase-defi60
cient DmMAEL mutants (K140A, K199A,
Q289A, D293A, D295A, D314A, andK328A) in mael-depleted OSCs and then40
monitored the levels of TE derepression.
Like wild-type DmMAEL, all seven of the20
ssRNase-deficient mutants rescued TE
derepression (Figures 4A and S4C). Since0
the ECHC-motif mutants of DmMAEL
Rescue
EGFP
FL-DmMael
DmMAELplasmidwere not expressed in E. coli as soluble
proteins, we could not examine their
ssRNase activities in vitro. Overall, these(Figure 3A). The H329A mutant retained the ssRNase activity, results suggested that the ssRNA cleavage activity of Maelindicating that His329 is not involved in ssRNA cleavage appears dispensable for Piwi/Mael-mediated TE silencing in(Figure 3B). Thus, despite our numerous attempts, the RNA Drosophila OSCs.
cleavage mechanism remains to be elucidated. Nonetheless,our mutational analyses support the notion that Mael does not DISCUSSION
share an active site with either RNase T1 or the DEDDh family
The present crystal structure revealed that DmMAEL adopts
members.
an RNase H-like fold but does not share catalytic residues
with the RNase H-like superfamily members. Unexpectedly,
The ssRNase Activity of Mael Appears Dispensable forour biochemical analyses revealed that DmMAEL has ssRNase
Piwi/Mael-Mediated TE Silencing in Drosophila OSCs
To examine the contribution of the MAEL domain to Piwi/ activity. We further showed that BmMAEL and MmMAEL also
Mael-mediated TE silencing, we overexpressed DmMAEL, have the ssRNase activities, and we identified seven potential
FL-DmMael, the four ECHC-motif single mutants (E131A, ssRNA-binding residues of DmMAEL. These results strongly
C288A, H291A, and C300A) of FL-DmMael, the ECHC-motif support our surprising finding that the MAEL domain posquadruple mutants (E131A/C288A/H291A/C300A) of FL- sesses the ssRNase activity, although it lacks the canonical
DmMael, and DmMAEL in mael-depleted OSCs and then moni- active site conserved across the RNase H-like superfamily
tored the expression levels of TEs by qRT-PCR. FL-DmMael and nucleases.
Previous studies showed that Mael participates in piRNA
DmMAEL rescued the derepression of a variety of somatic TEs(mdg1, 297, blood, Tabor, gypsy, and ZAM) (Figures 4A, 4B, biogenesis in germ cells of the fly ovary and mouse testis (Limand S4A), indicating that the MAEL domain plays a central and Kai, 2007; Sienski et al., 2012; Aravin et al., 2009; Castañedarole in Piwi/Mael-mediated TE silencing in Drosophila OSCs, et al., 2014). In the adult mouse testis, a ribonucleoprotein comconsistent with a previous report (Sienski et al., 2012). All of plex comprising Mael, the PIWI protein MIWI, and the Tudorthe ECHC-motif mutants of FL-DmMael and DmMAEL failed domain-containing protein TDRD6 is involved in the processing
to rescue TE derepression (Figures 4A and S4B), highlighting of precursor transcripts into mature pachytene piRNAs, a classthe functional significance of the ECHC motif for Piwi/Mael- of mammalian piRNAs (Castañeda et al., 2014). Notably, thenuclease activity of MIWI is not required for piRNA biogenesis
mediated TE silencing.
Cell Reports 11, 1–10, April 21, 2015 ª2015 The Authors 7

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030(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 ofMael is 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
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.
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,
8 Cell Reports 11, 1–10, April 21, 2015 ª2015 The Authors(2) its endogenous ssRNA substrates, and (3) the physiologicalrelevance of its ssRNase activity.
EXPERIMENTAL PROCEDURES
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).
ACCESSION NUMBERS
The atomic coordinates of DmMAEL have been deposited in the Protein
Data Bank under accession number 4YBG. The sequencing data of Bombyx
mori Mael have been deposited in GenBank under accession number
LC032360.
SUPPLEMENTAL INFORMATION
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.
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) (toO.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

Please cite this article in press as: Matsumoto et al., Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor
Maelstrom, Cell Reports (2015), http://dx.doi.org/10.1016/j.celrep.2015.03.030

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