Cell Reports

ReportYb Integrates piRNA Intermediates
and Processing Factors into Perinuclear Bodies
to Enhance piRISC Assembly
Yukiko Murota,1,2,6 Hirotsugu Ishizu,3,6 Shinichi Nakagawa,4 Yuka W. Iwasaki,2 Shinsuke Shibata,5 Miharu K. Kamatani,2Kuniaki Saito,2 Hideyuki Okano,5 Haruhiko Siomi,2 and Mikiko C. Siomi3,*
1Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan
2Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
4RIKEN, Wako, Saitama 351-0198, Japan
5Department of Biomedical Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
6Co-first author*Correspondence: siomim@bs.s.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.celrep.2014.05.043
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARYPIWI-interacting RNAs (piRNAs) direct Piwi torepress transposons and maintain genome integrity
in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear
Yb bodies, the centers of piRNA biogenesis. Here,
we show that piRNA intermediates arising from thepiRNA cluster flamenco (flam) localize to perinuclear
foci adjacent to Yb bodies, termed Flam bodies.
RNAi-based screening of piRNA factors revealed
that Flam body formation depends on Yb, the corecomponent of Yb bodies, while Piwi and anotherYb body component, Armitage, are dispensable
for formation. Abolishing the RNA-binding activityof Yb disrupts both Flam bodies and Yb bodies.
Yb directly binds flam, but not transcripts fromneighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing
factors selectively into Flam bodies and Yb bodies,
respectively. We suggest that Yb is a key upstream
factor in the cytoplasmic phase of the piRNA
pathway in ovarian somatic cells.
INTRODUCTIONPIWI-interacting RNAs (piRNAs) are small noncoding RNAs of
23–30 nt that are enriched in animal germlines. piRNAs specifically interact with PIWI proteins to form piRNA-induced
silencing complexes (piRISCs) and direct them to repress transposons and thus maintain genome integrity in the gonads (Ishizu
et al., 2012; Juliano et al., 2011; Siomi et al., 2011). Loss-of-function mutations of PIWI proteins or piRNA biogenesis impairment
cause derepression of transposons, leading to defects in gametogenesis and sterility (Aravin et al., 2007; Khurana and Theurkauf, 2010).

Drosophila express three PIWI proteins: Piwi, Aubergine (Aub),and AGO3 (Siomi et al., 2011). In germ cells in the ovaries, primary piRNAs originate from intergenic piRNA clusters throughthe primary piRNA processing pathway in a Dicer-independent
fashion and are loaded onto Piwi and Aub. Following this, Piwi
localizes to the nucleus to mediate transposon silencing. In
contrast, Aub localizes to the cytoplasm, where it plays a rolein the piRNA amplification cycle cooperating with AGO3 throughreciprocal RNA cleavage that depends on PIWI-Slicer (endonuclease) activity (Brennecke et al., 2007; Gunawardane et al.,2007; Ishizu et al., 2012; Malone et al., 2009). In this system,transposon transcripts in both sense and antisense orientations
are consumed as piRNA precursors; thus, transposon silencing
and piRNA production occur simultaneously, enabling a constant supply of piRNAs in cells.
Somatic cells in ovaries express Piwi, but not Aub or AGO3;
therefore, they fail to amplify piRNAs. Thus, all piRNAs are primary and are specifically loaded onto Piwi (Ishizu et al., 2012).
The Piwi-piRNA complex is then translocated to the nucleus, inwhich it implements transcriptional silencing in cooperation
with cofactors such as Maelstrom and DmGTSF1/Asterix (Dönertas et al., 2013; Muerdter et al., 2013; Ohtani et al., 2013;
Sienski et al., 2012). Whether Piwi in germ cells analogously collaborates with Maelstrom and DmGTSF1/Asterix to achievetransposon repression remains unknown.
Investigation of the primary piRNA pathway using a cultured
Drosophila ovarian somatic cell (OSC) line and fly ovaries has revealed that perinuclear Yb bodies are the centers for piRNA processing and piRISC formation in ovarian somatic cells (Olivieri
et al., 2010; Saito et al., 2010). Protein constituents of Yb bodies
include Yb, Armitage (Armi), Shutdown (Shu), Sister of Yb (SoYb)
and Vreteno (Vret), all of which contain domains associated withRNA metabolism; for instance, Yb shows significant similarity to
DEAD-box proteins and contains a Tudor (Tud) domain, whereas
Vret contains two Tud domains and an RNA-recognition motif(Ishizu et al., 2012). Armi belongs to the Upf1p family of ATPdependent RNA helicase (Cook et al., 2004). Loss of Yb bodycomponents prevents accumulation of primary piRNAs in the
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(legend on next page)104 Cell Reports 8, 103–113, July 10, 2014 ª2014 The Authorssoma; thus, they are all required for primary piRNA biogenesis
and gonadal development (Haase et al., 2010; Handler et al.,2011; Olivieri et al., 2010, 2012; Qi et al., 2011; Saito et al.,2010; Szakmary et al., 2009; Zamparini et al., 2011).
Nascent, piRNA-unloaded Piwi in OSCs interacts with Armiand Yb, and the resultant complex associates with piRNA intermediates in Yb bodies (Olivieri et al., 2010; Saito et al., 2010).
Depletion of Yb disrupts Yb bodies, liberating other components
into the cytosol. Under these conditions, Piwi and Armi still associate, although the heterodimer does not contain piRNA intermediates. As a result, piRISC formation fails. It is likely that Yb is at
the top of the hierarchy for Yb body formation and piRISC formation and that Armi, although categorized as an RNA helicase
based on peptide sequence similarity, binds RNA substrates(piRNA intermediates) upon localization to Yb bodies.
Zucchini (Zuc), a phospholipase D superfamily member, isa single-strand-specific endonuclease required for converting
piRNA intermediates to mature piRNAs (Ipsaro et al., 2012; Nishimasu et al., 2012). Depletion of Zuc in OSCs results in dispersalof Yb bodies, because piRNA-unloaded Piwi is stalled and fails
to localize to the nucleus (Saito et al., 2010). Without Zuc,an excess of unprocessed piRNA intermediates accumulate
in OSCs as ribonucleoprotein (RNP) complexes with Armi, Yb,
and Piwi; as a result, few piRNAs are produced (Haase et al.,2010; Nishimasu et al., 2012; Saito et al., 2010). These defects
can be rescued by ectopic expression of wild-type (WT) Zuc,but not by ectopic expression of endonuclease-deficient Zucmutants (Nishimasu et al., 2012). Zuc has a mitochondrial targeting signal at the N terminus, and indeed mouse Zuc (also known
as MitoPLD) localizes to the outer membranes of mitochondria,
facing into the cytosol in mammalian cells (Choi et al., 2006).
Zuc signals in OSCs can be detected in close proximity to Yb
bodies (Saito et al., 2010), as pi-bodies and piP-bodies, which
are considered to be the sites of piRNA biogenesis in mouse
testis, are located in intermitochondrial regions (Pillai and
Chuma, 2012). This type of intracellular architectural arrangement might raise the rates of Zuc-mediated conversion of piRNAintermediates to mature piRNAs in the cells.
The functions of piRNA protein factors have been well studied.
In contrast, the cell biology of piRNA precursors is poorly understood. Therefore, in this study, we performed RNA-fluorescencein situ hybridization (RNA-FISH) in OSCs to visualize RNA transcripts arising from the flamenco (flam) piRNA cluster. flam is
the main source of primary piRNAs in OSCs and somatic follicle
cells in Drosophila ovaries (Brennecke et al., 2007; Malone et al.,2009; Saito et al., 2009). A P-element insertion in flam causes
derepression of transposons such as gypsy and piRNA loss inmutant follicle cells (Brennecke et al., 2007; Mével-Ninio et al.,2007). RNA-FISH showed that flam transcripts are condensed
into perinuclear foci adjacent to Yb bodies, which we termed
Flam bodies. Recently, a similar type of body named ‘‘Dot
COM’’ was reported (Dennis et al., 2013). The similarities and differences between Flam bodies and Dot COM will be described
below. RNAi-based screening of piRNA factors revealed that
Flam body formation depends on Yb, the core component of
Yb bodies, while Piwi and another Yb body component, Armi,
are dispensable for the assembly. Depletion of Zuc, whichcauses excessive accumulation of unprocessed piRNA intermediates (Haase et al., 2010; Nishimasu et al., 2012), results in thedispersion of Flam bodies, which overlap considerably with Yb
bodies. Abolishing the RNA-binding activity of Yb disrupts both
Flam bodies and Yb bodies. Yb directly binds flam transcripts,
but not the transcripts of a neighboring protein-coding gene,
DIP1. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies,
respectively. We propose that Yb is a key upstream factor in
the cytoplasmic phase of the piRNA pathway in the ovarian
soma.
RESULTSflam Transcripts Are Localized to Perinuclear Flam
Bodies in Ovarian Soma
To examine the cellular localization of flam transcripts in OSCs,
we performed RNA-FISH using a flam-specific riboprobe complementary to the 50 region of the flam transcript (Figure 1A).
This region produces a substantial amount of primary piRNAs
in OSCs (Saito et al., 2010) (Figure 1A). By RNA-FISH, perinuclear, punctate signals were observed, suggesting that theflam-piRNA precursors/intermediates concentrate at the cytoplasmic bodies (Figures 1B and S1A). A recent report showedthat flam/COM transcripts (flam is also known as COM) are enriched in a single nuclear focus, termed Dot COM (Dennis et al.,2013). However, using electron microscopy in situ hybridization(EM-ISH), we detected flam signals in the cytoplasm (Figures
1C and S1B). Fluorescence quantification analysis revealed
that the average number of Flam bodies per cell was 1.5 (Figure 1B). Signals from RNA-FISH riboprobes that were usedoriginally by Dennis et al. (COM 508 and 527) (Dennis et al.,2013) coincided with the flam signals (Figure S1C). Hereinafter,

Figure 1. Flam Bodies Localize Adjacent to Yb Bodies in Drosophila Ovarian Soma

(A) Schematic drawing of the genomic flam locus and an upstream protein-coding gene DIP1 on chromosome X. Mature piRNAs uniquely mapping to the 50 regionof the flam locus corresponding to 21,506,000 to 21,510,000 on chromosome X (Saito et al., 2009) are shown by blue bars. A 583 nt riboprobe for RNA-FISH
complementary to the flam transcripts is shown by a magenta box (probe) (chrX: 21,506,472–21,507,054).
(B) Flam body (green) and Yb body (magenta) in OSCs visualized by RNA-FISH and immunofluorescence using anti-Yb antibody, respectively. The nucleus isstained with DAPI (blue). Scale bar, 2 mm. The numbers of Flam bodies and Yb bodies were determined by quantification analysis of flam and Yb signals in OSCs(n = 20).
(C) Electron microscope in situ hybridization using the probe shown in (A) detects a flam signal (white arrowhead) in the cytoplasm of OSC. The far-right image isan enlarged image of the middle image (partial). A white dot line shows where the nucleus is. Scale bar, 2 mm.
(D) Immunoelectron microscopy using anti-Yb antibody shows a perinuclear Yb body in an OSC (white arrowhead). The right-hand image is an enlarged image ofthe left-side image (partial). m, mitochondria. Scale bar, 2 mm.
(E) Flam body (green) and Yb bodies (magenta) in follicular cells of Drosophila ovaries. The nuclei are stained with DAPI (blue). Scale bar, 2 mm.
(F) The flam loci (green) in the nucleus and Yb body (magenta) in the cytoplasm. The nucleus is stained with DAPI (blue). Scale bar, 2 mm.

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we refer to the flam-positive, perinuclear bodies as Flambodies.
Flam bodies appear to be similar to Yb bodies, both in size andnumber. Therefore, we set out to understand the spatial relationship between Flam bodies and Yb bodies in OSCs by combining
flam RNA-FISH and immunofluorescence using an anti-Yb antibody that we raised in this study (Figure S1D). Immunoelectron
microscopy using the antibody confirmed the perinuclear localization of Yb bodies (Figure 1D). Flam bodies were frequently
located adjacent to Yb bodies and tended to be closer to the nucleus than Yb bodies (Figures 1B and S1A). The average numberof Yb bodies was similar to that of Flam bodies (1.4 per cell)(Figure 1B). Both signals were also detected in the somatic follicle cells of Drosophila ovaries (Figures 1E and S1E). Thus, they
are not specific for cultured cells. In flam mutant follicle cells,
Flam bodies were not detected, confirming the specificity of
the RNA-FISH probe (Figure S1F). EM-ISH confirmed the cytoplasmic localization of Flam bodies in follicle cells (Figure S1G).
DNA-FISH (Figure S1H) was conducted concomitantly withimmunofluorescence using the anti-Yb antibody. No spatial
correlation was found between flam nuclear foci and Yb bodies(Figures 1F and S1I).
Flam Bodies Are Not the Sites of Mature piRNA
Accumulation
It is possible that mature flam-piRNAs, rather than their intermediates and/or full transcripts, might be the major components ofFlam bodies. To examine this, we visualized Flam bodies and Yb
bodies in Piwi-depleted OSCs. Loss of Piwi decreased the levelof mature piRNAs drastically (Saito et al., 2009, 2010). This was
because Piwi is the sole protein loaded with mature piRNAs in
OSCs and, thus, piRNAs are destabilized without Piwi. If maturepiRNAs were the major RNA components of Flam bodies, Piwi
depletion would cause disappearance of these bodies. However, they were unaffected by Piwi depletion (Figure 2A). These
results indicate that Piwi is dispensable for Flam body formationand that Flam bodies are not the sites of mature piRNA storage.
piRNA Intermediates Concentrate in Flam Bodies
Depletion of Zuc caused Yb body dispersion and stalling of Piwi
at Yb bodies (Saito et al., 2010). This particular fraction of Piwi
was associated with few or no mature piRNAs, although theArmi complex containing Yb and Piwi still bound piRNA intermediates (Saito et al., 2010). These phenomena correlate well with
our recent finding that Zuc is the endoribonuclease necessary forconverting piRNA intermediates to mature piRNAs (i.e., piRNAmaturation) (Nishimasu et al., 2012). We asked if Flam body for-mation is affected by loss of Zuc. RNA-FISH in Zuc-depleted
OSCs showed that Flam bodies were dispersed similarly to
Yb bodies (Figure 2B). Interestingly, the two fluorescent signals
were superimposed (Figure 2B). We speculated that theincreased level of piRNA intermediates might have caused thesuperimposition. To examine this, northern blotting was performed to detect flam transcripts using probes designed torecognize the 50 end region of flam (the ‘‘probe’’ region in Figure 1A). In naive OSCs, a smeary signal for flam transcripts
was only slightly detected, although Flam bodies were clearlyvisible by RNA-FISH (Figures 2B and 2C). However, partial
flam transcripts, being several hundred to 4,000 nt in length,were aberrantly accumulated upon Zuc depletion (Figures 2C
and S2A) in agreement with our earlier observation (Nishimasu
et al., 2012). The corresponding signal decreased after treatmentof the cells with flam small interfering RNAs (siRNAs) targetingthe probed flam region (Figures 2C and S2A). These results
suggest that the smeared northern blot signal mostly reflects
flam-piRNA intermediates. After transfection of cells with flamsiRNAs, the fuzzy fluorescent signals of Yb bodies and Flambodies returned to normal, as in naive OSCs (Figure 2D). We
suggest that Flam bodies are where flam-piRNA intermediates
concentrate and that piRNA intermediates originating from lociother than flam would be minor. Treatment of normal OSCs
with flam siRNAs did not disrupt Flam bodies (Figure S2B) andbarely affected the levels of flam transcripts (Figure S2C),
although flam siRNAs effectively downregulated flam transcripts
in Zuc-depleted cells (Figure 2C). flam siRNAs were designed totarget the region corresponding to the flam RNA-FISH probe(Figure 1A). Thus, it is plausible that flam-piRNA intermediates
at Flam bodies would not be accessible to the RNAi machinery
under normal conditions likely due to the compactness of thebodies.
Yb Is Necessary for Formation of Both Yb Bodies andFlam Bodies
Depletion of Yb, but not other Yb body components, disrupts Yb
body formation (Handler et al., 2011; Saito et al., 2010). We
examined if depletion of Yb would affect Flam body formation.
Interestingly, neither Flam bodies nor Yb bodies were detectedin the cells (Figures 3A and S3A). Expression of siRNA-resistant
Yb (Yb WT-r) restored the formation of both structures (Figures
3A and S3A). Thus, Flam bodies depend on Yb for their formation
as do Yb bodies. Yb mutant follicle cells contain neither Flambodies nor Yb bodies (Figure S3B). Depletion of Armi and Vret,other components of Yb bodies (Olivieri et al., 2010), affected
neither Flam nor Yb body formation (Figure S3C and data not

Figure 2. Depletion of Zuc, but Not of Piwi, Affects Yb Body and Flam Body Formation(A) Western blotting shows the efficiency of Piwi depletion. Piwi siRNA (siPiwi) has little effect on Flam body (green) and Yb body (magenta) formation. Enhanced
GFP siRNA (siEGFP) was used as a negative control. Nuclei are stained with DAPI (blue). Scale bar, 2 mm.
(B) Transfection of OSCs with Zuc siRNA (siZuc) led to superimposition of Flam bodies (green) with Yb bodies (magenta). Nuclei are stained with DAPI (blue). Scalebar, 2 mm.
(C) Northern blotting shows that transfection of OSCs with Zuc siRNA (siZuc) induces aberrant accumulation of flam-piRNA intermediates in OSCs. Transfectionof Zuc-depleted OSCs with flam siRNAs (siflam) targeting the probed flam region downregulates the expression of flam-piRNA intermediates. Enhanced GFPsiRNA (siEGFP) was used as a negative control. RpL5 mRNA was visualized as a loading control.
(D) Flam bodies (green) and Yb bodies (magenta) in Zuc-depleted OSCs. Either enhanced GFP siRNA (siEGFP) or flam siRNA (siflam) was transfected into OSCs.
Nuclei are stained with DAPI (blue). Scale bar, 2 mm.

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Figure 3. Yb Requirement for Flam Body and Yb Body Formation(A) Depletion of Yb in OSCs abolishes formation of Flam bodies (green) and Yb bodies (magenta). Expression of siRNA-resistant Yb (Yb WT-r) restored theformation of both structures. Nuclei are stained with DAPI (blue). Scale bar, 2 mm.
(B) The RNA-binding activity of Yb is abolished by either the Q399A or the D537A mutation in the Yb NTD. Western blotting using the anti-Yb antibody shows thatan approximately equal amount of Yb was obtained through Yb-CLIP (top). However, the intensity of the Yb Q399A- and D537A-RNA complexes was much lower
than that of the Yb P455A- and Yb WT-r-RNA complexes (bottom).

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108 Cell Reports 8, 103–113, July 10, 2014 ª2014 The Authors

shown). These observations support the notion that Yb is thecentral player in piRNA biogenesis in OSCs; namely, Yb triggers
the fabrication of two perinuclear structures, Yb bodies and Flambodies, the hubs for piRNA maturation and piRNA intermediateconcentration, respectively, in the piRNA pathway.
RNA Binding of Yb through the N-Terminal RecA-like
Domain Is Required for Yb Body and Flam Body
Formation
Yb shows significant similarity to DEAD-box RNA helicases such
as Vasa in Drosophila and DDH5 and DDX18 in humans, especially in the N-terminal RecA-like domain (NTD) consisting of a
Q motif and motifs I, Ia, Ib, II, and III (Figure S3D). To examine
the functional involvement of the NTD of Yb in the piRNA
pathway, we individually mutated highly conserved residues in
Yb, Gln399, Pro455, and Asp537, to Ala. Gln399, Pro455, andAsp537 reside within the Q motif, motif Ia, and motif II, respectively (Figure S3D). In Vasa, Gln272 in the Q motif and Asp399
in motif II, which correspond to Gln399 and Asp537 in Yb, areinvolved in ATP binding, while Pro326 in motif Ia, which corresponds to Yb-Pro455, contributes to RNA substrate binding(Sengoku et al., 2006). ATP binding by Vasa is necessary for
RNA binding (Banroques et al., 2010). Thus, we expected thatthe alteration of Gln399, Pro455, and Asp537 in Yb to Ala would
abolish the RNA-binding function of Yb. Indeed, crosslinking andimmunoprecipitation (CLIP) showed that mutation of Gln399
and Asp537 to Ala (Q399A and D537A, respectively) severely
decreased the RNA-binding activity of Yb (Figure 3B). Thus, Yb
is a bona fide RNA-binding protein and binds RNA substrates
through the conserved NTD.
To determine if the RNA-binding activity of Yb is required in thepiRNA pathway, three mutants of siRNA-resistant Yb (Q399A,
P455A, and D537A), as well as Yb WT, were expressed in Ybdepleted OSCs (Figure S3E). Northern blotting showed that while
the WT control Yb WT-r rescued the defect in piRNA accumulation caused by loss of Yb function, the Q399A and D537A
mutants (Q399A-r and D537A-r) failed to rescue the defective
phenotype (Figure 3C). The P455A mutant (P455A-r) behaved
similarly to the WT control (Figure 3C), suggesting that Pro455,
despite its high conservation (Figure S3D), is dispensable forpiRNA biogenesis. We also examined the expression level ofthe mdg1 transposon in the transfected cells, and found thatYb WT-r and P455A-r rerepressed mdg1, but Q399A-r and
D537A-r failed to do so (Figure 3D). Q399 and D537, but not
P455, in the Yb NTD are necessary for both piRNA production
and piRNA-mediated transposon silencing.
We then asked if Yb WT-r and the three Yb mutants in Ybdepleted OSCs were able to restore formation of Flam bodies
and Yb bodies. Yb WT-r and P455A-r were able to form Yb
bodies, while Q399A-r and D537A-r were dispersed in thecytosol and did not accumulate at specific foci (Figures 3E
and S3F). Correlating with this, Flam bodies appeared when

Yb WT-r and P455A-r were expressed (Figures 3E and S3F).
However, the expression of Q399A-r and D537A-r did not resultin the formation of Flam bodies (Figures 3E and S3F). Quantitative RT-PCR (qRT-PCR) detected flam-piRNA intermediates in
Q399A-r- and D537A-r-expressing OSCs, where endogenous
Yb had been depleted by RNAi (Figure S3G). Thus, the substitution of endogenous Yb with Q399-r or D537A-r mutant did notinterfere with flam expression.
Yb Directly Binds flam-piRNA Intermediates, but Not
Neighboring Protein-Coding DIP1 Transcripts
Does endogenous Yb in OSCs indeed bind flam transcriptsthat serve as piRNA intermediates in piRNA biogenesis? To
address this question, HITS-CLIP was performed in OSCs using
an anti-Yb antibody. Illumina HiSeq2000 sequencing resulted ina total of 72,464,026 reads, consisting of 353,894 unique reads.
We mapped these unique reads to the Drosophila genome, and220,197 reads (62.2%) were mapped to a unique position. Annotation of mapped Yb-CLIP tags was similar to that of Piwi-associated piRNAs (Saito et al., 2009); over half of the reads (50.3%)
were mapped to transposon regions (Figure S4A).
Further analysis of the Yb-CLIP tags showed that Yb most
preferably binds transcripts from the flam locus among all
piRNA clusters (Figure S4B). We then precisely analyzed the
Yb-CLIP tags mapped to the flam locus. The distribution of
the Yb-CLIP tags on the flam locus revealed that Yb in OSCs
indeed associates with flam transcripts (Figure 4A). The YbCLIP tags significantly overlapped with flam-piRNAs associatedwith Piwi in OSCs (Figure 4A). By contrast, none of the Yb-CLIPtags were mapped to a neighboring coding gene, DIP1 (Figures
4A and 4B), which is highly expressed in OSCs (Sienski et al.,2012; Cherbas et al., 2011). Yb-CLIP tag mapping to proteincoding genes that produce genic piRNAs (Saito et al., 2009)
revealed that Yb almost exclusively binds the 30 UTR, but not
the protein coding sequence (CDS) or 50 UTR of the transcripts(Figure S4C). The 30 UTR, but not the CDS or the 50 UTR, of
genic piRNA-producing mRNAs serves as the piRNA sources(Robine et al., 2009; Saito et al., 2009). These results suggestthat Yb directly, and somewhat selectively, binds piRNA intermediates in OSCs.
DISCUSSION
In this study, we visualized flam-piRNA intermediates in OSCs
and follicle cells using RNA-FISH and EM-ISH and revealed
that they concentrate at perinuclear Flam bodies. Flam bodies
locate in very close proximity to Yb bodies, the sites of piRNAmaturation and piRISC formation. We postulated that flam signals might also be detectable within Yb bodies. However, thiswas not the case (Figures 1B and S1A). The simplest explanation
for this observation is that piRNA processing at Yb bodies occurs
so quickly, and the processed piRNAs localize to the nucleus as(C) Expression of Yb WT-r and P455A-r, but not Q399A-r and D537A-r, rescued the defect in piRNA accumulation in Yb-depleted OSCs. A piRNA arising from a
traffic jam (tj) gene (Saito et al., 2009) was visualized by northern blotting using a specific DNA probe. miR310, loading control.
(D) Expression of Yb WT-r and P455A-r rerepressed the mdg1 transposon in Yb-depleted OSCs, but expression of Q399A-r or D537A-r could not.
(E) Mutation of Q399A and D537A in Yb abolished Flam body (green) and Yb body (magenta) formation in OSCs. Endogenous Yb was depleted by RNAi. Nuclei
are stained with DAPI (blue). Scale bar, 2 mm.

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110 Cell Reports 8, 103–113, July 10, 2014 ª2014 The Authors(A) Mapped Piwi-piRNA reads and Yb-CLIP tags on the DIP1
coding and flam cluster region. Forward reads are shown inblue, and reverse reads are shown in red. The number on theside of tracks denotes the number of reads in each position.
Genomic coordinates are marked above the tracks. The bottom
panel shows a further zoom-in view of sequences around theregion where the RNA-FISH probe was designed to bind.
(B) A further zoom-in view of sequences of the DIP1 gene in (A)(boxed by a dotted blue line).
(C) A new model for primary piRNA biogenesis in OSCs. The
transcription and nuclear export of the transcripts are considered to be the ‘‘nuclear phase’’ of piRNA biogenesis. The flamtranscripts become flam-piRNA intermediates by ‘‘shortening’’
in either the nucleus or the cytoplasm. The mechanism underlying the shortening process remains unknown. Yb binds piRNAintermediates in the cytoplasm and locates to Flam bodies.
piRNA intermediates are further processed to mature piRNAs
at Yb bodies with the help of Zuc on mitochondria (MITO) andloaded onto Piwi, giving rise to the piRISC. The piRISC isimported to the nucleus, where the RNP complex exerts its
function: transcriptional silencing of transposons. Yb-piRNAintermediate association, piRNA maturation, and piRISC assembly are considered to be the ‘‘cytoplasmic phase’’ of piRNAbiogenesis. Components of Yb bodies include Yb, Armi, Vret,SoYb, and Shu, all of which are required for piRNA biogenesis
and piRISC formation in OSCs.

piRISCs so immediately, that the flam signal at Yb bodies was
below the level of detection at Yb bodies.
In Zuc-depleted cells, flam transcripts were detected predominantly as flam-piRNA intermediates, being several hundred to
4,000 nt in length (Figures 2C and S2A), while the full transcriptional unit of flam is estimated to be over 180 kb (Brennecke
et al., 2007; Malone et al., 2009). Both Yb body and Flam bodyformation require Yb, or more precisely, its RNA-binding activity
through its NTD (Figures 3B, 3E, and S3F). Yb binds flam-piRNAintermediates directly (Figure 4A). Based on these findings, we
propose a new model for primary piRNA biogenesis in ovariansoma (Figure 4C), in which the association of Yb with piRNA intermediates, which most likely occurs in the cytoplasm because
Yb is a cytoplasmic protein (Olivieri et al., 2010; Saito et al.,2010), is the initiation point of the cytoplasmic phase of piRNAbiogenesis. This follows the nuclear phase of piRNA biogenesis:
flam transcription and nuclear export of flam transcripts throughthe nuclear pores. flam transcription is initiated by RNA polymerase II and requires the transcriptional factor Cubitus interruptus (Goriaux et al., 2014). However, it is not known by which
export factors and in what lengths the flam transcripts areexported from the nucleus. Further investigation will be required
for a detailed understanding of the nuclear phase of piRNAbiogenesis.
The locations of the genomic flam loci in the nucleus andFlam bodies do not seem to be arranged to be close to eachother, meaning that the flam transcripts move a long distanceto arrive at Flam bodies (Figure 4C). Do the flam transcripts
move within the nucleus to get closer to Flam bodies before
export to the cytoplasm? Alternatively, does nuclear export
occur first and then flam transcripts are localized to Flambodies? Yb localization in the cytoplasm seems to be so dynamic that a point mutation in Yb that disrupts the RNA-binding
capacity of Yb drastically changes the subcellular localizationof Yb, causing it to be scattered evenly in the cytosol (Figures
3E and S3F). Thus, the latter scenario appears more likely, inwhich Yb plays a crucial role; upon nuclear export, Yb captures
flam transcripts through direct binding and localizes them, as
flam-piRNA intermediates, to Flam bodies. Flam body formation depends on the RNA-binding activity of Yb, a cytoplasmic
protein (Olivieri et al., 2010; Saito et al., 2010); this notion
further supports the idea that Flam bodies are cytoplasmic
structures.
Unlike flam transcripts, DIP1 mRNAs were virtually undetectable in Yb-CLIP tags (Figures 4A and 4B), although the DIP1
protein-coding gene and the flam piRNA cluster are neighbors
on chromosome X (Figure 1A) and DIP1 is expressed in OSCs.
We looked carefully at the sequences of Yb-CLIP tags but foundno obvious consensus sequences. Yb may recognize binding
substrates owing to higher-order structures.
Immunoelectron microscopy using an anti-Yb antibody
showed that Yb bodies are often attached to mitochondria, towhich Zuc endoribonuclease, the piRNA intermediate processor, anchors on the surface to face into cytoplasmic Yb bodies(Figure 1D). This peculiar spatial arrangement of Zuc andYb bodies, along with Flam bodies, integrates all the ingredients
necessary for primary piRNA production locally, enhancing
the rates of piRISC assembly. Another virtue of this perinuclear

arrangement is that it enables assembled piRISCs to be
immediately imported into the nucleus, where the RNP complex (i.e., the PIWI-piRNA complex) exerts its nuclear-specificfunction of silencing transposon transcription (Sienski et al.,2012). How does Yb decide where within the perinuclear
region to integrate all the materials necessary for primarypiRNA biogenesis? Reconstitution of the whole machinery in,for instance, nongonadal somatic Schneider2 cells, in which
no primary piRNAs are otherwise expressed, might address
this fundamental question.
EXPERIMENTAL PROCEDURES
Detailed procedures are provided in Supplemental Experimental Procedures.
Drosophila Strains
Yellow white (y w) and the flam [KG00476] and the fs(1)Yb[72] alleles wereused. Fly stocks were maintained at 25 C.
Cell Culture and RNAi
OSCs were grown in OSC medium prepared from Shields and Sang M3 Insect
Medium (Sigma) supplemented with 0.6 mg/ml glutathione, 10% fetal bovine
serum, 10 mU/ml insulin, and 10% fly extract. RNAi was performed using
RNA oligos shown in Table S1.
Production of Anti-Yb Antibodies
Monoclonal antibodies against Yb were raised primarily as described previously (Ohtani et al., 2013). A recombinant protein consisting of glutathione
S-transferase and the N-terminal region of Yb (200 amino acids) was purified
from E. coli and injected into mice.
RNA-FISH
The fluorescein isothiocyanate (FITC)- and digoxigenin-labeled RNA probes
were prepared using RNA labeling mixture (Roche) and SP6RNA polymerase(Roche) according to the manufacturer’s instructions. To prepare a probe specific for the flam locus, OSC genomic DNA was used as a template for PCR.
The primers used for PCR are indicated in Table S1. In situ hybridization
was carried out essentially as described previously (Sone et al., 2007).
DNA-FISH
Digoxigenin- or biotin-labeled DNA probes were prepared using Nick Translation Mix (Roche) according to the manufacturer’s instructions. To prepare theprobes, bacterial artificial chromosome clones DME1-021J16 (upstream of theflam locus) and DME1-014M21 (downstream of the flam locus), were used as
templates. OSCs were treated with ice-cold 0.75 M KCl for 5 min and then,after resuspending in acetic acid-methanol (1:3), spread onto slides. The slides
were treated essentially according to the procedures of Masumoto et al.
(Masumoto et al., 1989).
Immunofluorescence
Immunofluorescence was performed primarily as described previously (Ohtani
et al., 2013; Saito et al., 2009).
Body Counting
Confocal images of immunofluorescence were transferred to the Columbus
System (PerkinElmer Japan) and analyzed by Building Block (PerkinElmer
Japan). Nuclei were masked and then perinuclear signals for Yb bodies andFlam bodies were detected.
Electron Microscopy In Situ Hybridization
OSCs were fixed with 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) overnight and then washed with RNase-free PBS. The samples were hybridized
with FITC-conjugated specific RNA probe, except that 0.5% Triton X-100
was used for 5 min. Samples were incubated with a primary rabbit anti-FITC(1:500) antibody and then washed in 0.1 M phosphate buffer (PB) containing

Cell Reports 8, 103–113, July 10, 2014 ª2014 The Authors 111

0.005% saponin. Samples were incubated for 24 hr at 4 C with nanogold-conjugated anti-rabbit secondary antibody (1:100, Invitrogen).

by a Grant-in-Aid for the Global COE program from MEXT to Keio University(H.S. and H.O.).

Immunoelectron Microscopy
OSCs were fixed with 0.1% glutaraldehyde and 4% PFA in 0.1 M PBS, followed by incubation with 5% Block Ace containing 0.1% saponin in 0.1 M
PB. The cells were stained with anti-Yb antibody (1:250) and nanogold-conjugated anti-mouse secondary antibody (1:100, Invitrogen).

Received: December 16, 2013
Revised: April 9, 2014
Accepted: May 21, 2014
Published: June 19, 2014
REFERENCES

Western Blot Analysis
Western blotting was performed primarily as described previously (Miyoshi
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Northern Blot Analysis
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Bioinformatic Analysis
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ACCESSION NUMBERS
Yb-CLIP tag sequencing data have been deposited in the Gene Expression
Omnibus under the accession number GSE54875.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,four figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2014.05.043.
AUTHOR CONTRIBUTIONS
M.Y., H.I., S.N., Y.W.I., S.S., M.K.K., and K.S. designed and performed experiments and helped write the manuscript. H.O. supervised electron microscopic
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ACKNOWLEDGMENTS
We thank H. Masumoto for technical advice and Y. Iyoda, H. Kotani, T. Yano,
and T. Nagai for technical assistance. We also thank M. Isogai and M. Shimura(PerkinElmer Japan) for their assistance with fluorescence quantification. This
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