Letterdoi:10.1038/nature25788

Hierarchical roles of mitochondrial Papi and
Zucchini in Bombyx germline piRNA biogenesis
Kazumichi M. Nishida1*, Kazuhiro Sakakibara1*, Yuka W. Iwasaki2, Hiromi Yamada1, Ryo Murakami1, Yukiko Murota1,
Takeshi Kawamura3,4, Tatsuhiko Kodama4, Haruhiko Siomi2 & Mikiko C. Siomi1

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs thatbind to PIWI proteins to control transposons and maintain genome
integrity in animal germ lines1–3. piRNA 3′ end formation in thesilkworm Bombyx mori has been shown to be mediated by the 3′-to-5′
exonuclease Trimmer (Trim; known as PNLDC1 in mammals)4,
and piRNA intermediates are bound with PIWI anchored onto
mitochondrial Tudor domain protein Papi5. However, it remains
unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr)3′-to-5′ exonuclease, both of which have pivotal roles in piRNAbiogenesis in Drosophila6–8, are required for piRNA processing inother species. Here we show that the loss of Zuc in Bombyx had noeffect on the levels of Trim and Nbr, but resulted in the aberrant
accumulation of piRNA intermediates within the Papi complex, andthat these were processed to form mature piRNAs by recombinantZuc. Papi exerted its RNA-binding activity only when bound withPIWI and phosphorylated, suggesting that complex assembly
involves a hierarchical process. Both the 5′ and 3′ ends of piRNAintermediates within the Papi complex showed hallmarks of PIWI
‘slicer’ activity, yet no phasing pattern was observed in maturepiRNAs. The loss of Zuc did not affect the 5′- and 3′-end formationof the intermediates, strongly supporting the idea that the 5′ end of
Bombyx piRNA is formed by PIWI slicer activity, but independentlyof Zuc, whereas the 3′ end is formed by the Zuc endonuclease.
The Bombyx piRNA biogenesis machinery is simpler than thatof Drosophila, because Bombyx has no transcriptional silencing
machinery that relies on phased piRNAs.
piRNAs are produced through an intricate biogenesis pathway composed of the primary pathway, the amplification loop (also known as
the ping-pong cycle), and Zuc-dependent phasing1–3,6–12. To understand the mechanism that underlies the amplification machinery, thesilkworm ovary-derived, cultured cell line BmN4 has been used13–15.
BmN4 cells express two PIWI proteins, Siwi and Ago3. Siwi- andAgo3-bound piRNAs show strong nucleotide and strand biases(uracil at position 1 (1U) for the antisense strand, and adenine at position (10A) for the sense strand), and are complementary through 10
nucleotides from their 5′​end, known as the ping-pong signatures14,15.
Both Siwi and Ago3 are cytoplasmic: that is, silkworms rely on solely
post-transcriptional silencing to control transposons, unlike Drosophila
and mice, which repress transposons both transcriptionally andpost-transcriptionally1–3.
Papi16 in BmN4 cells is anchored to the surface of mitochondria
through a mitochondrial localization signal (MLS), and binds Siwi
and Ago3 through their symmetrically dimethylated arginine (sDMA)
residues5. Depletion of Papi was shown to have little effect on the levelsof piRNAs, although piRNAs became several bases longer exclusively attheir 3′​ end5. Trim was identified as the 3′​-to-5′​exonuclease requiredfor piRNA 3′​processing in Bombyx4.

Zuc endonuclease is necessary for piRNA biogenesis, particularly
for phased piRNA production in Drosophila and mice6–8,17–19. Nbrexonuclease functions in piRNA 3′​-end formation in Drosophila8,20.
The silkworm genome contains homologues of Zuc and Nbr genes(KAIKObase; http://sgp.dna.affrc.go.jp), both of which are expressedin BmN4 cells (Extended Data Fig. 1a, b). However, loss of Trim andNbr did not affect the levels of Flag-PIWI-loaded piRNAs, whereas
loss of Papi nearly completely abolished the PIWI–piRNA association(Fig. 1a and Extended Data Fig. 1a, c). Papi is essential for piRNA
production and formation of the piRNA-induced silencing complex(piRISC) in silkworm germline cells.
PIWI-loaded piRNAs in Trim-depleted cells seemed to be subtly
upshifted on RNA gels (Fig. 1a). The mean sizes of Siwi- and Ago3bound piRNAs were 28.2 and 27.6 nucleotides (in control cells), and 28.6
and 28.2 nucleotides (in Trim-knockdown cells), respectively (Fig. 1b);that is, Siwi- and Ago3-bound piRNAs produced with no Trim function
were on average 0.4 and 0.6 nucleotides longer than those in control cells.
piRNA sequencing confirmed these results (Extended Data Fig. 2a).
The 1U/10A and strand biases were greatly maintained after Trim
depletion (Extended Data Fig. 2b, c), suggesting that the changes inpiRNA size seem to be attributed to changes at the 3′​end. The population of piRNAs was also barely changed by Trim depletion (Extended
Data Fig. 2c). In addition, lack of Nbr caused no notable changes inSiwi- and Ago3-bound piRNAs (Extended Data Fig. 2a–c). Trim maythus act to fine-tune piRNA size at the 3′​ end.
piRNAs undergo 2′​-O-methylation by the methyltransferase Hen1and become resistant to β​-elimination21,22. Both Siwi- and Ago3-boundpiRNAs, even longer ones, showed firm resistance to β​-elimination,
regardless of the presence or absence of Trim (Fig. 1c and Extended
Data Fig. 3a). It seems that piRNA 2′​-O-methylation occurs irrespectiveof Trim depletion. This is at odds with a previous report that claims
that the lack of Trim impaired piRNA 3′​end formation, including
2′​-O-methylation, leading to a severe reduction in the piRNA level insilkworm cells4.
Mass spectrometric analysis of endogenous PIWI proteins in
BmN4 cells revealed that 11 and 5 arginine residues of Siwi andAgo3, respectively, were sDMA-modified (Extended Data Fig. 3b, c
and Supplementary Table 1). We substituted 9 out of the 11 a­ rginine­residues in Siwi to lysine residues (Siwi-9RK), which completely­abolished the Siwi–Papi association (Extended Data Fig. 4a). The Ago3
mutant, which should have lost its sDMA modification, failed to bindPapi, as has been reported previously5 (Extended Data Fig. 4a). Bothmutants were barely loaded with piRNAs (Extended Data Fig. 4b), andfailed to accumulate at nuage perinuclear foci23, the site for germline
piRNA biogenesis (Extended Data Fig. 4c). Thus, sDMA modification
is essential for the Papi association, nuage localization and piRISC formation of PIWI. Both Siwi and Ago3 were sDMA-modified even after1

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan. 2Department of Molecular Biology, Keio University School of Medicine, Tokyo
162-8582, Japan. 3Proteomics Laboratory, Isotope Science Center, The University of Tokyo, Tokyo 113-0032, Japan. 4Laboratory for Systems Biology and Medicine, Research Center for Advanced
Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
*These authors contributed equally to this work.
2 6 0 | N A T U R E | V O L 5 5 5 | 8 m ar c h 2 0 1 8© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Letter RESEARCH

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Figure 1 | Papi is essential for piRNA biogenesis and piRISC formationin BmN4 cells. a, Flag-tagged Siwi and Ago3 at the N termini (F-Siwi
and F-Ago3, respectively) are loaded with piRNAs in Nbr-knockdown(KD) and Trim-knockdown but not Papi-knockdown BmN4 cells. IP,
immunoprecipitation. b, piRNAs appear to be slightly longer when Trimis depleted. c, β​-elimination treatment of Siwi- and Ago3-bound piRNAs
from Trim-knockdown and control cells. d, RNAs within the Papi complex
and bound to Siwi were 32P-labelled at the 5′​end. ‘Isotype ctrl’ denotes
a non-immune IgG antibody. e, Wild-type (WT) Papi, but not the KH

mutant (mut) Papi, binds to RNAs. Papi-F, Flag-tagged Papi at the
C terminus. f, Myc–Siwi is unloaded with piRNAs when the KH mutant
is expressed in Papi-depleted cells. F-eGFP, Flag-tagged enhanced green
fluorescent protein. g, Papi does not bind RNAs in Siwi-depleted BmN4
cells. h, Papi is phosphorylated (Papi-P) in BmN4 cells. i, Model showingthat int-piRNA associates with the Papi–Siwi complex on mitochondria.
Siwi-sDMA and Papi phosphorylation (P) are required for the assembly.
Siwi may be replaced by Ago3 in this model. Tud, Tudor domain.

Papi depletion (Extended Data Fig. 4d), suggesting that the modification occurs before the Papi–PIWI association.
32
P-labelling of RNAs within the Papi complex revealed that the levelsof mature piRNAs in the complex were approximately 14.6% of thoseof piRNAs bound to Siwi itself (Fig. 1d), strongly supporting the ideathat the piRISC is displaced from Papi after the completion of piRISC
formation. The Papi complex contained not only Siwi but also Ago3 as
expected, but the level of Ago3 was estimated to be about 10% of thatof Siwi in the complex (Extended Data Fig. 5a).
The Papi complex contained RNA molecules longer than piRNAs(Fig. 1d), and this gave a positive result with a northern probe for
RT3-1 (Extended Data Fig. 5b). RT3-1 was one of the abundantpiRNAs loaded specifically onto Siwi14. piRNA intermediates (int-­
piRNAs) were detected similarly even after Siwi was forcibly displaced
from Papi (Extended Data Fig. 5c). The int-piRNAs are therefore­physically associated with Siwi in the Papi complex.

Papi contains two KH domains besides the Tudor domain and MLS(Fig. 1e). KH domains are RNA-binding motifs found in RNA-binding
proteins that function in various types of RNA metabolism24. However,whether Papi exhibits its RNA-binding activity through KH domains has
not been examined experimentally. Cross-linking immunoprecipitation(CLIP) experiments showed that Papi–Flag was efficiently cross-linked
with RNAs in BmN4 cells, as was endogenous Papi (Fig. 1e and Extended
Data Fig. 5d). The Papi–Flag KH mutant, in which Ile69 and Val142 weremutated to asparagine residues, failed to bind RNAs (Fig. 1e). These two
residues are highly conserved in KH domain-containing proteins andare crucial for the RNA-binding activity24. The Siwi–piRNA association
was impeded when the Papi mutant was expressed instead of endogenous
Papi (Fig. 1f and Extended Data Fig. 5e). Thus, the RNA-binding activityof Papi through KH domains is essential for piRISC formation.
Not only piRNAs but also int-piRNAs were hardly detected with the
Siwi-9RK mutant (Extended Data Fig. 5f). Also, endogenous Siwi in
8 m ar c h 2 0 1 8 | V O L 5 5 5 | N A T U R E | 2 6 1© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

RESEARCH Letter- piRNA- piRNA

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Figure 2 | Zuc is essential for piRNA biogenesis and piRISC formationin BmN4 cells. a, RNAs co-immunoprecipitated with Flag-Siwi and
Flag-Ago3 are visualized by 32P labelling. b, RT3-1 int-piRNAs aberrantly
accumulate in the Papi complex after Zuc depletion. c, Trim depletion
does not affect the levels of RT3-1 int-piRNAs in the Papi complex.
d, The presence of endogenous Siwi, Ago3, Papi, Trim and Zuc in the
mitochondrial (mito) fraction. Cyto, cytoplasmic fraction. Tubulin
was used as a loading control (bottom). e, The distribution patterns ofendogenous Siwi, Ago3, Papi, Trim and Zuc in mitochondrial fractions
1–19 separated by sucrose gradient sedimentation. Fraction 1 contains the
top (lightest) fraction. f, Zuc and Siwi are co-immunoprecipitated with
Papi from mitochondrial lysates.

Papi-depleted cells was not loaded with either piRNAs or int-­piRNAs(Extended Data Fig. 5g). Moreover, the intensity of the Papi CLIP
­signal decreased markedly when Siwi was depleted (Fig. 1g), althoughint-­piRNAs were detected similarly in total RNAs irrespective of thepresence of Siwi (Extended Data Fig. 5h). The Ago3–Papi association
should be maintained after Siwi depletion. However, the CLIP signal
was very low, agreeing with our earlier notion that the Ago3 level is low
in the complex. It is likely that int-piRNAs join the Papi complex only
after the Siwi–Papi association.
Both endogenous and exogenous Papi appeared as a doublet but only
the top band was cross-linked with RNAs (Fig. 1e, g and Extended Data
Fig. 5d). The top band does not represent a splicing variant because
ectopically expressed Papi from the full-length cDNA appeared asa doublet. However, Papi became a single band after phosphatase­treatment (Fig. 1h). Thus, Papi is subjected to phosphorylation andthis modification is essential for it to bind RNAs.
The findings suggest that the assembly of the Papi–PIWI–int-piRNAcomplex occurs via a hierarchical process (Fig. 1i), which would­possibly occur to ensure the funnelling of Siwi–piRNA intermediates toPapi–Siwi and Ago3–piRNA intermediates to the Papi–Ago3 complex.
If Papi binds int-piRNA first, there must be a high chance that both
Siwi and Ago3 would end up with the same set of piRNAs, disrupting
the piRNA amplification. We hypothesized that the regulation of Papi
phosphorylation is to keep Papi free from RNA until it associates with
PIWI. However, this seems to be unlikely, given that Siwi depletion had
a minimal effect on Papi phosphorylation (Fig. 1g).
Zuc depletion severely decreased the levels of Siwi- and Ago3-loaded
piRNAs (Fig. 2a and Extended Data Fig. 6a). In sharp contrast, RT3-1
piRNA intermediates strongly accumulated in the Papi complex after
Zuc depletion (Fig. 2b and Extended Data Fig. 5c). The depletion ofTrim did not lead to the accumulation of int-piRNAs (Fig. 2c andExtended Data Fig. 6b). The levels of Trim and Nbr (examined bywestern blotting and quantitative PCR, respectively) were unaffectedby Zuc loss (Extended Data Fig. 6b, c). Thus, Zuc is responsible forpiRNA processing in BmN4 cells.
Papi, Zuc and Trim are present in the BmN4 mitochondrial­fraction (Fig. 2d). In sucrose density-gradient experiments, Zuc andPapi were detected more strongly in lower-density fractions (Fig. 2e,­fractions 3–8), whereas Trim was mostly found in higher-density ones
(Fig. 2e, fractions 10–12). Zuc co-immunoprecipitated with Papi and
Siwi (Fig. 2f). The mitochondrial Siwi complex contained Papi and Zuc(Extended Data Fig. 6d). The interaction of Zuc with the Papi–Siwicomplex during the 3′​-end piRNA processing was suggested.
We sequenced the libraries generated from 65–100-nucleotide-longRNAs extracted from the mitochondrial Papi complex before and after
Zuc depletion (Extended Data Fig. 6e). More than 98% (98.09% in
control and 98.65% in Zuc knockdown) of the reads contained one ormore sequences of Siwi- or Ago3-bound piRNAs, indicating that thesequenced reads correspond to intermediate piRNAs. 1U/10A biases
were detected in the intermediate piRNA reads (Fig. 3a), suggestingthat piRNAs are generated from the 5′​end of the int-piRNA sequences.
More than half of the intermediate piRNAs had piRNAs aligned totheir 5′​end (51.94% for control and 59.58% for Zuc knockdown), suggesting that a large proportion of int-piRNAs produce piRNAs fromtheir 5′​end. Apparent phasing pattern was not detected within ­piRNAs
mapped to the same intermediate (Extended Data Fig. 6f), agreeing
with the previous report demonstrating that exogenous ­piRNAs
in BmN4 cells showed only weak phasing25. In Drosophila, phased
piRNAs are loaded onto Piwi and transcriptionally control transposons.
However, Bombyx lacks a Piwi homologue and relies solely on cytoplasmic PIWIs to silence transposons post-transcriptionally. Therefore, ourfinding that Bombyx produces no phased piRNAs appears reasonable.
We examined the distance from the 3′​end of each int-piRNA to the
5′​end of the next downstream intermediate, and found that the mostcommon 3′​-to-5′​distance was 1 nucleotide (Fig. 3b). This suggests thata single cleavage event, possibly by the Siwi or Ago3 slicer, produces
the 3′​end of one int-piRNA and the 5′​end of the adjacent downstream
int-piRNA, as in the case of the phased piRNAs. We then focused on
the cleavage site of two adjacent int-piRNAs and analysed the population of piRNAs that possess 10-nucleotide complementarity at thecleavage site (Fig. 3c). More than 94% (94.21% for control and 98.31%
for Zuc knockdown) of adjacent int-piRNA pairs had piRNAs that werecomplementary to 10 nucleotide from the 5′​end of the downstream
int-piRNA and the 3′​end of the upstream int-piRNA. In ­addition, we
calculated the proportion of int-piRNAs with complementary piRNAat each position from the 5′​end of the downstream int-piRNA, which
was found to be highest at the 10-nt position for both Siwi- and Ago3associated piRNAs (Fig. 3d). These results suggest that Siwi and Ago3
generate both 5′​and 3′​ends of int-piRNAs by slicer activity. Thiswas observed in both control and Zuc-depleted BmN4 cells (Fig. 3),
suggesting that Zuc is not involved in the production of both 5′​ and3′​ends of int-piRNAs.
Alteration of His169 at the active site of Drosophila Zuc to alanine
abolished its endonuclease activity18. Incubation of recombinant wildtype Bombyx Zuc, but not the corresponding mutant His141Ala, witha naked 50-nucleotide RNA (1U50) produced 7–31-nucleotide RNAs
dose-dependently (Fig. 4a and Extended Data Fig. 7a–c). Zuc may
preferentially cleave after cytosine, and given that the 3′​side of guanine tends to be avoided, A–C/A and U–C/U/G may rarely be cleaved(Extended Data Fig. 7c). The cleavage pattern of an 80-nucleotide RNA,a 30-nucleotide extended version of 1U50 at the 3′​end, was very similar
to that of 1U50 (Extended Data Fig. 7d, e). The predicted structuresof the two RNA molecules were different (data not shown), suggestingthat high-dimensional structures have a low effect on Zuc cleavage.
When RNA substrate was pre-loaded onto Flag-tagged Siwi, theproduct size was mostly in the range of 27–31 nucleotides (Fig. 4b
and Extended Data Fig. 7f, g), which are typical or permissible sizes

2 6 2 | N A T U R E | V O L 5 5 5 | 8 m ar c h 2 0 1 8© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Letter RESEARCHa2

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Figure 3 | Papi-associated int-piRNAs are generated by Siwi and Ago3
slicer. a, Nucleotide bias of 65–100-nucleotide Papi-associated int-piRNAs
in control and Zuc-depleted cells. b, Analyses of distance between Papiassociated intermediates in control and Zuc-depleted BmN4 cells. The
distance between the 3′​end of the upstream intermediate and the 5′​ endof the downstream intermediate on the same genomic strand is analysed.
c, Illustration showing the cleavage of adjacent intermediates by Siwi
or Ago3. The frequencies of the presence of a complementary piRNAwithin 10 nucleotides from the 5′​end of the downstream intermediate(piRNA intermediate 1) and 3′​end of the upstream intermediate(piRNA intermediate 2) are indicated for control and Zuc-knockdown
cells. d, Complementarity between piRNAs and adjacent intermediate
sequence pairs. Graphs indicate the relative frequencies of the presenceof a complementary piRNA (y axis), with the indicated distance at thedownstream intermediate (x axis).

for silkworm piRNAs14. Similar results were obtained when the Siwi–
RNA complex was pre-incubated with Papi (Fig. 4c and Extended Data
Fig. 7h). Wild-type Zuc also processed RT3-1 int-piRNAs within theendogenous Papi complex to mature RT3-1 piRNA (Fig. 4d). Thus,
Zuc endonuclease is the piRNA 3′​-end processing factor in silkworm
germline cells.
A new model for piRNA biogenesis in silkworm germline cells
is shown in Extended Data Fig. 8a. In Bombyx, Papi might bind

int-piRNAs towards the 3′​end, whereas the int-piRNA 5′​end is heldby Siwi, most likely inserted into the 5′​binding pocket26 (Extended
Data Fig. 8a, b). Under this structural arrangement, it would be nearly
impossible for Trim or Nbr to process int-piRNAs from the very 3′​
end to mature piRNAs because Papi acts as an obstacle to the 3′​-to-5′​
exonuclease reaction. Zuc is an endonuclease and so is able to process int-piRNAs even under such circumstances. Zuc shows only
a subtle nucleotide preference in RNA cleavage. This unique trait
helps the p­ rotein to act like an ‘exonuclease’, as a replacement of Nbrin Drosophila, to determine the length of piRNA in the biogenesis
pathway.
In mouse testes, the loss of the Papi homologue TDRKH (alsoknown as TDRD2) causes piRNA precursors to be accumulated on
PIWI, because without Papi, nucleases responsible for piRNA 3′​-end
formation, such as Zuc (known as MITOPLD in mice) and/or Trim,are incapable of processing the 3′​ end17,19,27,28. In flies, the loss of Papi
affected piRNA phasing, but hardly affected the levels of transposons ingerm cells6,29. The piRNA pathway is highly conserved among animal
species, but species diversity is evident from a mechanistic perspective.

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Figure 4 | Zuc processes int-piRNAs to mature piRNAs. a, Recombinant
wild-type Zuc, but not the catalytically inactive Zuc(H141A) mutant, cleaves
a naked 50-nucleotide RNA (1U50) in vitro. b, Wild-type Zuc cleaves 1U50
loaded onto Siwi. c, Wild-type Zuc cleaves 1U50 loaded onto the Papi–Siwicomplex. d, Wild-type Zuc processes RT3-1 int-piRNAs accumulated in the
Papi complex by Zuc depletion to mature RT3-1 piRNAs.

Online Content Methods, along with any additional Extended Data display items andSource Data, are available in the online version of the paper; references unique tothese sections appear only in the online paper.
received 12 June 2017; accepted 26 January 2018.
Published online 28 February 2018.
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11. Homolka, D. et al. PIWI slicing and RNA elements in precursors instructdirectional primary piRNA biogenesis. Cell Reports 12, 418–428 (2015).
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Supplementary Information is available in the online version of the paper.
Acknowledgements We are grateful to T. Mannen for preparing materials formass spectrometry, T. Suzuki for comments on our in vitro Zuc processing
assays and Y. Ono for support with the bioinformatics. We also thank S. Ohnishi
for technical assistance and other members of the Siomi laboratories fordiscussions and comments on the manuscript. This work was supported by
grants from the Ministry of Education, Culture, Sports, Science and Technologyof Japan to K.M.N., Y.W.I., Y.M., H.S. and M.C.S. R.M. is supported by CREST, theJapan Science and Technology Agency. T.Ka. was supported by grants fromthe New Energy and Industrial Technology Development Organization, Japan,
and Translational Systems Biology and Medicine Initiative from the Ministry of
Education, Culture, Sports, Science and Technology of Japan. T.Ko. is a recipientof Molecular Dynamics for Antibody Drug Development, First Program Grant
from the Japan Society of Promotion of Science.
Author Contributions K.M.N. generated monoclonal antibodies and performed
biochemical analyses of piRNAs, int-piRNAs and piRNA factors. K.S. carriedout in vitro experiments with help from R.M. H.Y. performed protein–protein
interaction analyses. Y.M. performed immunofluorescence analyses. Y.W.I.
performed bioinformatics analyses. T.Ka. and T.Ko. performed LC–MS/MS
analysis. M.C.S. designed the experiments with other authors, supervised anddiscussed the work, and wrote the manuscript. H.S. discussed and supervised
the study. All authors commented on the manuscript.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial
interests. Readers are welcome to comment on the online version of the paper.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations. Correspondence andrequests for materials should be addressed to M.C.S. (siomim@bs.s.u-tokyo.
ac.jp).
Reviewer Information Nature thanks J. Brennecke, S. Chameyron and the otheranonymous reviewer(s) for their contribution to the peer review of this work.

2 6 4 | N A T U R E | V O L 5 5 5 | 8 m ar c h 2 0 1 8© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Letter RESEARCH
Methods

Production of monoclonal antibodies. Monoclonal antibodies were produced
essentially as described previously14. Mice were immunized with bacterially
produced glutathione S-transferase (GST)-tagged Papi (amino acids 430–629),
GST–Trim (amino acids 1–240) or GST–Zuc (amino acids 63–206). The Papi, Trimand Zuc cDNAs corresponding to the particular regions were obtained by RT–PCR
using total RNAs from BmN4 cells. The PCR primers used are summarized in the‘Oligonucleotides’ section.
Plasmid construction. pIB-myc and pIB-3xFlag were generated using a pIB vector(Thermo Fisher Scientific). Vectors to express Myc–Siwi and Papi–Flag were generated by inserting Siwi and Zuc cDNAs into pIB-myc and pIB-3xFlag, respectively.
Vectors for expressing the Papi–Flag KH mutant, the Papi–Flag siRNA-resistantmutant, the Flag–Siwi RK mutant and the Flag–Ago3 RK mutant were generated
by inverse PCR using expression vectors for Papi–Flag (this study), Flag–Siwi14
and Flag–Ago314 as templates. The PCR primers used are summarized in the‘Oligonucleotides’ section.
RNAi and transgene expression. Double-stranded RNAs (dsRNAs) were­produced by in vitro T7 transcription, followed by annealing in water. The PCRprimers to generate dsRNAs are summarized in the ‘Oligonucleotides’ section.
BmN4 cells were transfected with 2 μ​g of dsRNAs (per 3 ×​  105 cells) using FuGENE
HD (Promega). To express proteins exogenously in BmN4 cells, the cells weretransfected with 2 μ​g of plasmids (per 6 ×​  105 cells) using FuGENE HD.
Western blot analysis. Western blotting was carried out as described ­previously14.
The Y12 antibody was a gift from G. Dreyfuss. Anti-HSP60 (StressMarq
Biosciences), anti-tubulin (Developmental Studies Hybridoma Bank), antiDDDDK-tag (Flag) (MBL), and anti-Myc (Sigma) antibodies were purchased.
qRT–PCR. qRT–PCR was performed as described previously30. The PCR primers
used are summarized in the ‘Oligonucleotides’ section.
Immunoprecipitation and RNA isolation. Immunoprecipitation of Flag–Siwi
and Flag–Ago3 was carried out essentially as described previously14. BmN4 wholeand mitochondrial lysates (Fig. 2c, f and Extended Data Fig. 6d) were preparedin b­ inding buffer (30 mM HEPES (pH 7.4), 150 mM potassium acetate, 5 mM
­magnesium acetate, 5 mM DTT, 0.5% Triton X-100, 2 μ​g ml−1 pepstatin, 2 μ​g ml−1
leupeptin and 0.5% aprotinin) and incubated with anti-Papi, anti-Siwi or anti-Flag
antibody bound to Dynabeads (Invitrogen) at 4 °C for 2 h. The beads were washedfour times with binding buffer. BmN4 whole (Fig. 1h and Extended Data Fig. 5b)
and mitochondrial (Extended Data Fig. 5c) lysates were prepared in ­binding buffer
containing 500 mM sodium chloride and incubated with anti-Papi or anti-Siwi
antibody. The beads were washed twice with binding buffer containing 500 mM
sodium chloride, and then twice with binding buffer. RNAs were eluted fromthe beads by phenol–chloroform after protease K treatment and precipitatedwith e­ thanol. RNA 32P-labelling14, β​-elimination22 and northern blotting12 werecarried out as described previously. RNAs were crosslinked to Hybond-N+ (GEHealthcare) by UV irradiation. The sequences of the RT3-1 probe and siRNA usedin the β​-elimination experiment are described in ‘Oligonucleotides’.
Generation of PIWI-associated small RNA libraries. Immunoprecipitation of
Flag–Siwi and Flag–Ago3 was carried out as described previously14. RNAs were
eluted from the immunoprecipitates by phenol–chloroform after protease K­treatment, and precipitated with ethanol. RNAs of 23–35 nucleotides in length
were eluted from the gels and used to generate small RNA libraries14.
Analysis of PIWI-associated small RNA sequences. Libraries were sequenced
using Illumina MiSeq (single-end, 51 cycles). For Siwi-associated small RNAs, atotal of 4,547,701 reads were obtained from the control sample, 3,054,386 reads
from the Trim-knockdown sample and 4,083,584 reads from the Nbr-knockdown
sample. For Ago3-associated small RNAs, a total of 3,855,720 reads were obtained
from the control sample, 3,702,496 reads from the Trim-knockdown sample and3,292,910 reads from the Nbr-knockdown sample. The analysis of small RNAs was
performed as described previously14. In brief, adaptor sequences were removed
from the obtained reads, and the reads in the range of 23–35 nucleotides wereused for further analysis (89–94% of the sequenced reads were in this size range).
The reads were mapped to the silkworm reference genome (downloaded from the
Silkworm Genome Research Program Database; http://sgp.dna.affrc.go.jp/data/
integretedseq.txt.gz) using Bowtie31, allowing no mismatches. Genome mapped
reads were extracted and aligned to 121 B. mori transposon consensus sequences(a gift from S. Kawaoka) using Bowtie31, allowing up to two mismatches. Using
transposon-mapped reads, the length distribution was calculated. Sequence logos
were calculated using the motifStack R package. The sequences were aligned to the
5′​end upon the calculation of sequence logos. Strand bias and frequency (reads per
million) of small RNA reads were calculated for 70 transposon consensus sequenceswith higher amount of mapped Siwi and Ago3 piRNAs (reads per million) in control
sample, and heat maps were depicted using Java TreeView software32.
Dephosphorylation treatment. Immunopurified Papi was incubated with
Antarctic Phosphatase (NEB) at 37 °C for 30 min for dephosphorylation.

Immunofluorescence. Immunofluorescence was carried out essentially as
described previously14. Anti-Flag antibody and Alexa Fluor 488 goat anti-mouse
IgG antibody (Invitrogen) were used as primary and secondary antibodies,­respectively.
Trypsin digestion and LC–MS/MS analysis. The method for trypsin digestionof protein has been described previously33. Liquid chromatography–tandem mass
spectrometry (LC–MS/MS) analysis was performed using an LTQ Orbitrap XLelectron transfer dissociation (ETD) mass spectrometer (Thermo Fisher Scientific).
The methods used for LC–MS/MS were slightly modified from those described
previously34. The mass spectrometer was operated in a data-dependent acquisition mode in which the mass spectrometry acquisition with a mass range of m/z
420–1,600 was automatically switched to MS/MS acquisition under the automatedcontrol of Xcalibur software. The top four precursor ions in an MS scan wereselected by Orbitrap, with resolution R =​ 60,000 and in subsequent MS/MS scansby ion trap in the automated gain control (AGC) mode in which the AGC values
were 5.00 ×​  105 and 1.00 ×​  104 for full MS and MS/MS, respectively. To analyse
dimethylation sites, ETD was used.
Database searching and protein identification. Database searches were­performed using the MASCOT 2.5.1 search engine (Matrix Science) against the
UniProtKB_2016-8 database (selected for B. mori), assuming trypsin as the digestion enzyme and allowing for trypsin specificity of up to four missed cleavages. The
database was searched with a fragment ion mass tolerance of 0.60 Da and a parent
ion tolerance of 3.0 p.p.m. The iodoacetamide derivative of cysteine was specified
as a fixed modification and methylation of arginine, oxidation of methionine,
dimethylation of arginine and acetylation of N termini were specified as variable
modifications. Scaffold (version Scaffold_4.7.5; Proteome Software) was used tovalidate MS/MS-based peptide and protein identifications. We accepted the ­peptide
identifications when the Peptide Prophet algorithm35 specified probabilities at>​95.0%. Sequence coverage was defined as the percentage of the protein in theidentified peptide sequence.
CLIP. CLIP was performed as described previously36. Dephosphorylation and
RNA radiolabelling with 32P were performed using T4 polynucleotide kinase.
Rescue assay. BmN4 cells were transfected with 500 pmol siRNA duplex(per 1 ×​  106 cells) using Cell Line Nucleofector Kit L (Lonza) and incubated at
27 °C for 72 h. The sequences of siRNAs are presented in ‘Oligonucleotides’. After
RNAi, cells were transfected with 2 μ​g of Papi–Flag plasmid using FuGENE HD
and incubated at 27 °C for 72 h. Cells were then transfected with 2 μ​g Myc–Siwi
plasmid and incubated at 27 °C for 24 h. BmN4 lysates were prepared in binding
buffer (30 mM HEPES (pH 7.4), 150 mM potassium acetate, 5 mM magnesium
acetate, 5 mM dithiothreitol (DTT), 0.1% Tergitol-type NP-40, 2 μ​g ml−1 pepstatin,
2 μ​g ml−1 leupeptin and 0.5% aprotinin) containing 500 mM sodium chloride, andincubated with anti-Myc antibody bound to Dynabeads. The beads were washedtwice with binding buffer containing 500 mM sodium chloride and then twicewith binding buffer. RNAs were eluted from the beads by phenol-chloroform after
protease K treatment and precipitated with ethanol. RNA radiolabelling was carriedout as described previously14.
Sucrose density gradient centrifugation. Mitochondria were prepared as
described previously37. The mitochondrial pellet was resuspended in gradientbuffer (30 mM HEPES (pH 7.4), 100 mM potassium acetate, 1 mM DTT, 4 mM
magnesium acetate, and 1% Tergitol-type NP-40), and centrifuged at 14,000g at
4 °C for 30 min. The supernatant was placed at the top of a 10–40% sucrose gradientand centrifuged in a Beckman MLS-50 rotor at 178,000g at 4 °C for 16.5 h.
Preparation of Papi-associated int-piRNA libraries. RNAs were eluted fromthe Papi immunoprecipitates by phenol–chloroform after protease K treatment
and precipitated with ethanol. RNAs were resolved by denaturing PAGE, and
65–100-nucleotide-long RNAs were eluted from gels. The libraries were generated
as described previously14.
Sequence analysis of Papi-associated int-piRNAs. Libraries were sequenced
using Illumina MiSeq (single-end, 111 cycles). A total of 10,584,873 reads wereobtained from the control sample and 7,932,439 reads were obtained from the Zucknockdown sample. Adaptor sequences were removed from the obtained reads,
and the reads were mapped to the silkworm reference genome (downloaded fromthe Silkworm Genome Research Program Database; http://sgp.dna.affrc.go.jp/
data/integretedseq.txt.gz) using Bowtie31, allowing no mismatches. Genome
mapped reads were extracted and aligned to B. mori transposon c­ onsensus
sequences using Bowtie31, allowing up to two mismatches. To reduce the effectof c­ ontaminant reads, the reads mapped to transposon consensus sequences wereused for ­further analysis. Sequence logos were calculated using the motifStack
R package (http://www.bioconductor.org/packages/release/bioc/html/motifStack.html). The sequences were aligned to the 5′​end upon the calculation ofsequence logos. Calculations of the distance between intermediates and piRNA-­
phasing analysis were performed as previously described6, and complementarity ofpiRNAs and adjacent intermediate pairs was calculated using an in-house script.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

RESEARCH Letter
The phasing analysis and piRNA-intermediate complementarity calculation were
performed using each pair of intermediates preserving the read count. Siwi andAgo3 piRNA sequences were obtained from previously published data14 (GEO
accession GSE58221).
Recombinant protein preparation. The cDNA encoding Zuc excluding MLS(residues 29–206) was cloned into pCold-GST vector. The Zuc(H141A) mutant
was generated from pCold-Zuc by inverse PCR. The protein was purified using
glutathione-Sepharose (GE Healthcare) with purification buffer (20 mM Tris-HCl(pH 8.0), 150 mM sodium chloride and 1 mM DTT). The proteins were treatedwith HRV3C protease (GE Healthcare) to remove the GST tag and further purified
by Enrich S (Bio-Rad). To yield pIZ-3xFlag-Siwi, 3xFlag-Siwi was amplified frompIB-3xFlag-Siwi14 by PCR and inserted into pIZ vector (Thermo Fisher). Flag–Siwi
was immunoisolated by ANTI-FLAG M2 Affinity Gel (Sigma) from BmN4 cells
and eluted by 500 ng μ​l−1 3×​Flag peptide (Sigma).
In vitro processing assay. The synthesized RNAs (GeneDesign) were 32P-labelled
at their 5′​ end. The sequences of RNAs are indicated in ‘Oligonucleotides;.
Radiolabelled int-piRNAs (104 c.p.m.) were incubated with 0.1 μ​g of recombinant
Zuc in 20 μ​l of buffer A18 at 26 °C for 30 min. A total of 1 μ​l of 1 μ​M radiolabelled
int-piRNAs and 200 ng of purified Flag–Siwi were mixed at 26 °C for 30 min in
20 μ​l of the loading buffer (30 mM HEPES (pH 7.4), 100 mM potassium acetate,
2 mM magnesium acetate, 20 mM creatine monophosphate, 1 mM ATP, 0.15 U μ​l−1creatine phosphokinase, 1 mM DTT and 0.5 U μ​l−1 RNasin (Promega)). Afterloading, the Flag–Siwi-containing mixture was incubated with Dynabeads (withanti-Siwi antibody or Papi immunoprecipitation product) at 4 °C for 1 h. The beads
were then washed five times with binding buffer. RNAs were isolated from thebeads with phenol–chloroform and precipitated with ethanol. They were thenresolved by denaturing PAGE. Immunopurified Papi complexes were incubatedwith 1 μ​g of recombinant Zuc in 30 μ​l of buffer A18 containing 2.5 mM EGTA and
0.1 U μ​l−1 RNasin at 27 °C for 0–180 min. RNAs were isolated from the beads withphenol–chloroform and precipitated with ethanol, after which they were transferred to Hybond-N (GE Healthcare). Next, RNAs were chemically crosslinked tomembrane using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as described
previously14. The sequence of probe RT3-1 is indicated in the ‘Oligonucleotides’
section.
Oligonucleotides. Primers used for producing constructs for Myc–Siwi, Papi–
Flag, siRNA-resistant Papi, Papi KH mutant, Flag–Siwi RK mutant, Flag–Ago3
RK mutant and pIZ-3xFlag-Siwi are listed below.
Myc-L (left, forward): 5′​ - TTCGAATTTAAAGCTCACCATGGGAGAGCAGAAACTGATC-3′​, Myc-R (right, reverse): 5′​-CTGCAGGAATTCGAT
CCGGGTACCAAGCTTGCTAG-3′​; 3xFlag-L: 5′​-GGCCCGCGGTTCGAAGACTACAAAGACCAT-3′​, 3xFlag-R: 5′​-AGTCAGATAAACTCAGATATCCTTGTCATC-3′​; Myc–Siwi-L: 5′​ - TGCAGCCCAGCGCTGGATCCATG
TCAGAACCGAGAGGTAG-3′​, Myc–Siwi-R: 5′​-CGAACCGCGGGCCCTTTAGAGGAAATATAAAGTTT-3′​; Papi–Flag-L: 5′​-TCGAATTTAAAGCTTATGTCATTGAACACAAAATT-3′​, Papi–Flag-R: 5′​-GAACCGCGGGCCCTCCTTTTCAAAAGCGGACTTAC-3′​; Papi siRNA-res-L: 5′​-CACCCAAGTCAACAGATAAAGTTGCTGAGCA-3′​, Papi siRNA-res-R: 5′​-ACTCCACTTTGACGCGAGCGCCACTGCGAT-3′​; Papi KH I69N mut-L: 5′​-TCCAACAACAAGGACCTTCAGAAGAAATCT-3′​, Papi KH I69N mut-R: 5′​-GCCATTGCGACCAATCAGAGCTGGAACAAT-3′​; Papi KH V142N mut-L: 5′​-GAGAACAACAATGATATTAGCCATCGCAGT-3′​, Papi KH V142N mut-R: 5′​-ACCTCCAGATCCAATTATTCTCCCACAAGA-3′​; Siwi RK mut1-L: 5­′​-­C­AG­AA­CC­GA­GA­GG­TA­AA­G
G­AA­AA­GC­TA­AA­GG­AA­AG­GC­TG­GT­AAGGGTGGTGATGGAGGC-3′​,­ Siwi
RK mut1-R: 5′​-­G­CC­TC­CA­TC­AC­CA­CC­CT­TA­CC­AG­CC­TT­TC­CT­TT­AG­CT­TT­
TC­CTTTACCTCTCGGTTCTG-3′​; S­iw­i RK mut2-L: 5′​-­C­GT­AG­TT­GG­CA­AG­
GG­CT­CT­AA­AA­AA­GG­GG­GT­GG­AAAAGTCCTTCCTG-3′​, Siwi RK mut2-R:
5′​-CAGGAAGGACTTTTCCACCCCCTTTTTTAGAGCCCTTGCCAACTACG-3′​;
Ago3 RK mut1-L: 5′​-CCAGGCAAAGGCAAGGGGAAAAGCTTAGCCC-3′​,
Ago3 RK mut1-R: 5′​-GGGCTAAGCTTTTCCCCTTGCCTTTGCCTGG-3′​; Ago3
RK mut2-L: 5′​-GTATAGGCGGTAAAGGAAAGGCAGCAGCATTG-3, Ago3
RK mut2-R: 5′​-CAATGCTGCTGCCTTTCCTTTACCGCCTATAC-3′​; Ago3
RK mut3-L: 5′​-CAGCTGGAACTGGAAAGGGATTCAAATTGC-3′​Ago3 RK
mut3-R: 5′​-GCAATTTGAATCCCTTTCCAGTTCCAGCTG-3′​; 3xFlag-Siwi-L:
5′​-TCGAATTTAAAGCTTCACCATGGACTACAAAGACCATGACGGTG-3′​,
3xFlag-Siwi-R: 5′​ - GAACGAGAAACGTAAAAGTTTAGAGGAAATATA
AAGTTTCATTC-3′​.
Primers used for producing constructs for GST–Papi, GST–Trim and GST–Z­uc are:
GST–Papi-L: 5′​-­T­GG­GA­TC­CC­CG­AA­TT­CA­AG­GA­CA­AA­GAGATACCTGG-3′​,
GST–Papi-R: 5­′​-GGCCGCTCGAGTCGACTCACTTTTCAAAAGCGGACT-3′​;
GST–Trim-L: 5′​-TGGGATCCCCGAATTCATGGATATCACCAAAGAAAA-3′​,
GST–Trim-R: 5′​ - GGCCGCTCGAGTCGACTTAGTTATCTTCCAGTA

TTGCTA-3′​; GST–Zuc-L: 5′​-TACCCTCGAGGGATCCACAAAAAGCATG
GAC-3′​, GST–Zuc-R: 5′​-CGACAAGCTTGAATTTTAACTGGTTATTGG-3′​.
Primers used for producing constructs for pCold-Zuc and the Zuc(H141A)
mutant are: pCold-Zuc-L: 5′​-TACCCTCGAGGGATCCAAGAAGAAGAAAGAA-3′​, pCold-Zuc-R: 5′​-CGACAAGCTTGAATTTTAACTGGTTATTGG-3′​;
Zuc(H141A)-L: 5′​-GCCCACAAGTTCTGCATAATAGATG-3′​, Zuc(H141A)-R:
5′​-CATTAGATTTGTAGACTTCATCCAG-3′.
Primers used to generate templates for dsRNA production are: T7dsLuc-L: 5′​-TAATACGACTCACTATAGGGGGAGAGCAACTGCATAAGGC-3′​,
T7-dsLuc-R: 5′ ​ - TAATAC GACTCACTATAGGGTC C CTATC GAAGGACTCTGG-3′​; T7-dsPapi-L: 5′​ - TAATACGACTCACTATAGGGTCTAGTGACATTCCGTGGTA-3′​, T7-dsPapi-R: 5′​-TAATACGACTCACTATAGGGAGCACTCCATGGCCTGGAAC-3′​; T7-dsTrim-L: 5′​-TAATACGACTCACTATAGGGTTCAGAAGTTTCAAAATGGT-3′​, T7-dsTrim-R: 5′​-TAATACGAC
TCACTATAGGGAGCGAAGAATTCATACAAAT-3′​; T7-dsZuc-L: 5′​-TAATACGACTCACTATAGGGATGGCAGTAACTCTTAGTAA-3′​ , T7-dsZuc-R:
5′​-TAATACGACTCACTATAGGGGTCCAGTTCAATGACCCTGC-3′​; T7-dsNbr-L:
5′​-TAATACGACTCACTATAGGGACAAATTGTTATGGCATTGG-3′​, T7-dsNbr-R:
5′​-TAATACGACTCACTATAGGGCAATCCATTTCAAGGCTTCA-3′​; T7-dsSiwi-L:
5′​-TAATACGACTCACTATAGGGGCAAATAATGTCAAATACCC-3′​, T7-dsSiwi-R:
5′​-TAATACGACTCACTATAGGGACCACCATCAGTGACGGCAG-3′.
Sequences of siRNA duplexes used for RNAi (DNA: normal font, RNA:
underlined) are: Luc siRNA duplex: 5′​-CGUACGCGGAAUACUUCGATT-3′​
and 5′​-UCGAAGUAUUCCGCGUACGTT-3′​; Papi siRNA duplex: 5′​-GGUCGAAAGUCCUAAAAGUTT-3′​and 5′​-ACUUUUAGGACUUUCGACCTT-3′.
Sequence of RT3-1 probe used for northern blot is: 5′​-ACCAGCCGATCGTC
ATCGCATCCGTTTA-3′. Sequence of siRNA used for β​-elimination is: 5′​-UGGUCUGCCUAAAGGUGUCGCUCUGC-3′. Primers used for qPCR are: Nbr-L:
5′​-ACAGCCAGTTCAAAATAGTTATTGC-3′​, Nbr-R: 5′​-TTGACCACAGTATTCACACAGAACT-3′​; Rpl3-L: 5′​-GGTGTCACCAAGGGCAAAGG-3′​,
Rpl3-R: 5′​-AGGATGCCAAGCTCCAATGC-3′.
Sequences of RNA used for processing assays are: 1U29: 5′​ - UCAAAAACUAACGGAUUGGUUUCGAACAG-3′​; 1U50: 5′​-UCAAAAACUAACGGAUUGGUUUCGAACAGUCACCCGCCCGGACAGGUCCC-3′​; 1U80: 5­′​-­UCAAAAACUAACGGAUUGGUUUCGAACAGUCACCCGCCCGGACAGGUCCC
CUACCUGUCCCUAAUCAACUGGCAGCCGGG-3′.
Statistics and reproducibility. Experiments in Figs 1a, e, 2b–e and 4d were performed three times independently with similar results. Experiments in Figs 1b–d,f–h, 2a, f and 4a–c were performed twice independently with similar results.
Experiments in Extended Data Figs 1b, c, 4b, 5b, c, f, g, 6a, b were performed threetimes independently with similar results. Experiments in Extended Data Figs 3a,4a, c, d, 5a, d, e, h, 6d and 7a–h were performed twice independently with similar
results. Experiments in Extended Data Fig. 3c were performed once. No statisticalmethods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments andoutcome assessment.
Data availability. Gel source images for Figs 1a–h, 2a–f and 4a–d and Extended
Data Figs 1b, c, 3a, 4a, b, d, 5a–h, 6a, b, d and 7a–h are available in Supplementary
Fig. 1. All other data supporting the findings of this study are available from thecorresponding author upon reasonable request. All sequencing data that support
the findings of this study were deposited in the NCBI Gene Expression Omnibus(GEO) with the GEO series accession number GSE107371.
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32. Saldanha, A. J. Java Treeviewextensible visualization of microarray data.
Bioinformatics 20, 3246–3248 (2004).
33. Fujinoki, M. et al. Identification of 36-kDa flagellar phosphoproteins associatedwith hamster sperm motility. J. Biochem. 133, 361–369 (2003).
34. Fujii, K. et al. Fully automated online multi-dimensional protein profilingsystem for complex mixtures. J. Chromatogr. A 1057, 107–113 (2004).
35. Keller, B. O., Wang, Z. & Li, L. Low-mass proteome analysis based on liquid
chromatography fractionation, nanoliter protein concentration/digestion, andmicrospot matrix-assisted laser desorption ionization mass spectrometry.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782, 317–329 (2002).
36. Murota, Y. et al. Yb integrates piRNA intermediates and processing factors into
perinuclear bodies to enhance piRISC assembly. Cell Rep. 8, 103–113 (2014).
37. Wieckowski, M.R., Giorgi, C., Lebiedzinska, M., Duszynski, J., & Pinton, P.
Isolation of mitochondria-associated membranes and mitochondria fromanimal tissues and cells. Nat. Protocols 4, 1582–1590 (2009).

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Letter RESEARCH

Extended Data Figure 1 | Production of monoclonal antibodies against
Papi, Trim and Zuc, and analysis of depletion upon RNA interference
treatment. a, Quantitative PCR with reverse transcription (qRT–PCR)
shows that Nbr was efficiently depleted by RNA interference (RNAi) in
BmN4 cells. Data are mean ±​ s.e.m. of three independent experiments.
b, Western blotting shows the specificity of anti-Papi, anti-Trim andanti-Zuc monoclonal antibodies raised in this study. HSP60 and tubulin
were used as loading controls. The images show the domain structuresof Papi, Trim and Zuc. Underlines indicate the antigen regions used for
producing the monoclonal antibodies. c, Western blotting shows that Papi
and Trim were efficiently depleted by RNAi in BmN4 cells.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

RESEARCH Letter

Extended Data Figure 2 | Siwi/Ago3-associated small RNAs upon Trimor Nbr depletion. a, Length distribution of transposon-mapped Flag-Siwiand Flag-Ago3-associated piRNAs. piRNAs appear to be slightly longer
when Trim was depleted. b, Sequence logos showing unaffected levels of

1U and 10A under Trim- or Nbr-depleted conditions. c, Strand bias andfrequency of piRNAs mapped to each transposon consensus sequence.
Depletion of Trim or Nbr has little effect on strand bias or the frequency ofpiRNAs mapped to each transposon consensus sequence.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Letter RESEARCH

Extended Data Figure 3 | sDMA modification of Siwi and Ago3.
a, A synthesized short interfering RNA (siRNA) (26 nucleotides) was
downshifted by β​-elimination, indicating that this siRNA is not 2′​-Omethylated. b, The amino acid sequences of the N-terminal regions ofwild-type Siwi, the Siwi-9RK mutant, wild-type Ago3 and the Ago3-5RK
mutant are shown. Arginine residues shown in red were determined to be

sDMAs in BmN4 cells. Arginine residues mutated to lysines areshown in green. c, Representative ETD tandem mass spectra for Siwi andAgo3 peptides, which include arginine modifications. Ac, acetylation;
Di, demethylation; Me, monomethylation. Charge, m/z and Mascot scoreare shown on the top right of each spectrum. All identified Siwi and Ago3
peptides are listed in Supplementary Table 1.

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RESEARCH Letter

Extended Data Figure 4 | Analysis of Siwi and Ago3 mutants. a, Wildtype Flag–Siwi and Flag–Ago3, but not Flag–Siwi-9RK and Flag-Ago35RK mutants, are co-immunoprecipitated with Papi from BmN4 cells.
b, Wild-type Flag–Siwi and Flag–Ago3, but not Flag–Siwi-9RK and Flag–
Ago3-5RK mutants, are loaded with piRNAs in BmN4 cells. The middle(sDMA) shows that neither the Flag–Siwi-9RK nor Flag–Ago3-5RK

mutant reacts with the Y12 antibody, which specifically recognizes sDMA.
c, Wild-type Flag–Siwi and Flag–Ago3, but not Flag–Siwi-9RK and
Flag–Ago3-5RK mutants, are localized to nuage in BmN4 cells (shown ingreen). Blue (DAPI staining) indicates the location of the nucleus. Scalebars, 10 μ​m. d, Papi depletion has little effect on sDMA modification of
Flag–Siwi and Flag–Ago3 expressed in BmN4 cells.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Letter RESEARCH

Extended Data Figure 5 | Papi complex analysis. a, Top, Flag–Siwi and
Flag–Ago3 expressed in BmN4 cells were immunoisolated with anti-Flag
antibody and probed with anti-Flag antibody after sequential dilution.
Bottom, Flag–Siwi and Flag–Ago3 immunoisolated from BmN4 cells (thesame samples as in the top panel) were probed with anti-Siwi and antiAgo3 antibodies, respectively. Siwi and Ago3 co-immunoprecipitated with
Papi were simultaneously probed with anti-Siwi and anti-Ago3 antibodies,
respectively. The Papi complex was equally divided into two fractions
and each fraction was used for each blot. Examination of the signal
intensity revealed that the amount of Siwi within the Papi complex wasapproximately equal to 1/1.6 volume of Flag–Siwi and that the amount of
Ago3 within the Papi complex was approximately equal to 1/16 volume ofFlag–Ago3. Comparison of the signal intensity on the top and bottom blots
suggests that the ratio of abundance of Siwi and Ago3 in the Papi complex
is 10:1. b, Northern blotting shows that the Papi complex contains RT3-1

int-piRNAs. c, Northern blotting shows that Siwi in a form associated with
Papi on mitochondria binds RT3-1 int-piRNAs independently of Papi.
The Siwi–int-piRNA association is maintained even after Zuc depletion.
d, CLIP analysis shows that only the long form, but not the short form,of endogenous Papi in BmN4 cells interacts with RNA in vivo. e, Western
blotting using anti-Papi (top) and anti-Flag (second from the top)
antibodies shows that wild-type Papi–Flag and the KH mutant are equally
expressed in BmN4 cells, in which endogenous Papi has been depleted byRNAi. Western blotting using anti-Myc (third from the top) shows thatthe levels of Myc–Siwi are approximately equal in the cells. Tubulin wasused as a loading control (bottom). Both wild-type Papi–Flag and the KH
mutant were mutated to be RNAi resistant. f, Flag–Siwi-9RK and Flag–
Ago3-5RK mutants bind with little int-piRNA. g, Flag–Siwi binds withlittle int-piRNA in Papi-lacking BmN4 cells. h, Northern blotting showsthat int-piRNAs are still present in Siwi-depleted BmN4 cells.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

RESEARCH Letter

Extended Data Figure 6 | RNAi efficiency, Siwi–Papi–Zuc interaction
and analysis of Papi-associated intermediates and piRNA phasing.
a, Western blotting shows that Zuc is efficiently depleted by RNAi. HSP60
is used as a loading control. b, Western blotting shows that Zuc and Trimare efficiently depleted by RNAi. Zuc and Trim depletion had little effecton the protein levels of Trim and Zuc, respectively. HSP60 is shown as aloading control. c, qRT–PCR shows that the level of Nbr is not affected by

Zuc depletion in BmN4 cells. Data are mean ±​ s.e.m. of three independent
experiments. d, Zuc and Papi are detected in the Siwi complex
immunoisolated from the mitochondrial fraction of BmN4 cells. e, The
size distribution of Papi-associated intermediates mapped to transposons.
f, Analyses of phased piRNAs in Papi-associated intermediates. The
distance between the 3′​end of the upstream piRNA and the 5′​end of thedownstream piRNA on the same genomic strand is analysed.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Letter RESEARCH

Extended Data Figure 7 | Analyses of Zuc RNA cleavage. a, Coomassie
brilliant blue (CBB)-stained gel showing purified wild-type Zuc and
the Zuc(H141A) mutant. b, Wild-type Zuc in a cleaves 1U50 in a dosedependent manner. c, Detailed analyses of Zuc RNA cleavage. The left gelshows RNA ladders ranging from 14 to 50 nucleotides. The right gel shows
RNA ladders ranging from 7 to 15 nucleotides. Relatively ‘strong’ RNAbands are indicated by red arrowheads (s1–s7). ‘Intermediate’ RNA bands
are indicated by blue arrowheads (i1–i11). Relatively ‘weak’ RNA bands
are indicated by grey arrowheads (w1–w7). 1U29 is an authentic RNA, thesequence of which is identical to that of 1U50 RNA over 1–29 nucleotides
from the 5′​end. Classification of strong, intermediate and weak RNA
bands is carried out in accordance with the intensity of each band.
d, An 80-nucleotide RNA, 1U80, is cleaved by wild-type Zuc.
e, Cleavage patterns of 1U50 and 1U80 by wild-type Zuc are compared.
f, CBB-stained gel showing purified Flag–Siwi. g, 1U80 pre-loaded onto
Flag-Siwi in f is cleaved by wild-type Zuc. h, The Siwi–1U80 RNA complex
was first incubated with Papi, which was immunopurified using an antiPapi antibody, and then treated with wild-type Zuc.

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

RESEARCH Letter

Extended Data Figure 8 | A new model for piRNA biogenesis in
Bombyx. a, A model for the ping-pong cycle in Bombyx. Papi is localizedon the surface of mitochondria through MLS, whereas KH domains are
required for Papi to exhibit RNA-binding activity. The Papi Tudor domain
and sDMA modification of Siwi and Ago3 are required for the Siwi–Papi
and Ago3–Papi interactions. It remains unclear how Papi is maintainedin an RNA-free state before the Papi–PIWI association. Also, it remains
unclear how piRISC upon its formation is displaced from Papi, and how
piRISC avoids re-association with Papi. Conformational change of piRISC
may be involved. b, Drosophila phased piRNA biogenesis involves
Zuc–Zuc endonucleolytic cleavage for piRNA 5′​and 3′​end formation.
PIWI-slicer-Nbr exonucleolytic trimming and PIWI-slicer-Zuc

endonucleolytic cleavage produce piRNAs in the ping-pong cycle. The roleof Drosophila Papi remains under discussion. Its functional homologue(s)(shown as X and Y) may function with Zuc and Nbr. Bombyx lacks a gene
homologue of Drosophila Piwi, so it does not have to accommodate phased
piRNA biogenesis. Because of this, Zuc endonuclease might not be used
for piRNA 5′​end formation. However, PIWI-slicer-Zuc endonucleolytic
cleavage produces piRNAs in the ping-pong cycle. We infer that the
3′​-to-5′​exonuclease cannot trim the 3′​end of the intermediate because
Papi impedes this reaction. This model shows that Bombyx piRNAs areproduced in a manner that depends on PIWI-slicer and Zuc. There may
also be alternative pathways that have less of an effect on the overall levelsof piRNA production.

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Initial submission

Revised version

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1. Sample size
Describe how sample size was determined.

Not applicable

2. Data exclusions
Describe any data exclusions.

nature research | life sciences reporting summary

Corresponding author(s): Mikiko C. Siomi

We had one data as “data not shown” in the manuscript.
The predicted structures of 80-nt and 50-nt RNAs are not similar to each other(page14): Because we thought that it was not crucial to show. However, we are
happy to show the data upon request.

3. Replication
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All attempts at replication were successful.

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Not applicable

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Describe whether the investigators were blinded togroup allocation during data collection and/or analysis.

Not applicable

Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

6. Statistical parameters
For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the
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The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same
sample was measured repeatedly
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The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more
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June 2017

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Clearly defined error bars
See the web collection on statistics for biologists for further resources and guidance.

1Policy information about availability of computer code

7. Software
Describe the software used to analyze the data in thisstudy.

For analyzing the reads obtained from deep sequencing analysis, cutadapt, Bowtie,
motifStack from R package, and in-house script for calculation of the distance
between intermediates and piRNA-phasing analysis (which is available uponrequest), were used.

For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be madeavailable to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance forproviding algorithms and software for publication provides further information on this topic.

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unique materials or if these materials are only available
for distribution by a for-profit company.

No unique materials were used.

nature research | life sciences reporting summary` Software

9. Antibodies
Describe the antibodies used and how they were validated Anti-Siwi, Ago3, PAPI, Zuc and Trim antibodies were produced from immunized
for use in the system under study (i.e. assay and species). mouse, respectively. Anti-myc (catalog number: C3956), Anti-HSP60 (catalog
number: SMC-110B), Anti-DDDDK-tag (Anti-Flag antibody) (catalog number: FLA-1)
and anti-Tubulin antibodies (catalog number: E7) were purchased from Sigma,
StressMarq Biosciences, MLB and DSHB, respectively. Y12 antibody was gifted from
Dr. Gideon Dreyfuss.

10. Eukaryotic cell lines`

a. State the source of each eukaryotic cell line used.

BmN4 cells were gifted from National Institute of Agrobiological Sciences (NIAS).

b. Describe the method of cell line authentication used.

We think that BmN4 cells used have been authenticated, because BmN4 were
gifted from NIAS.

c. Report whether the cell lines were tested for
mycoplasma contamination.

BmN4 cells were not tested for mycoplasma contamination.

d. If any of the cell lines used are listed in the databaseof commonly misidentified cell lines maintained by
ICLAC, provide a scientific rationale for their use.

BmN4 cells are not misidentified cell lines.

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June 20172