RNA assignment

10.1101/gad.1968110Access the most recent version at doi: 2010 24: 2499-2504 originally published online October 21, 2010Genes Dev.

Astrid D. Haase, Silvia Fenoglio, Felix Muerdter, et al.

Drosophilapathway in Probing the initiation and effector phases of the somatic piRNA

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RESEARCH COMMUNICATION

Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila Astrid D. Haase,1 Silvia Fenoglio,1 Felix Muerdter,1

Paloma M. Guzzardo,1 Benjamin Czech,1

Darryl J. Pappin,1 Caifu Chen,2 Assaf Gordon,1

and Gregory J. Hannon1,3

1Watson School of Biological Sciences, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA; 2Genomic Assays R&D Life Technologies, Foster City, California 94404, USA

Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.

Supplemental material is available at http://www.genesdev.org.

Received July 9, 2010; revised version accepted September 17, 2010.

Eukaryotic small RNAs regulate gene expression through various mechanisms, intervening at both transcriptional and post-transcriptional levels (for review, see Ghildiyal and Zamore 2009). Small RNAs are divided into classes according to their mechanism of biogenesis and their particular Argonaute protein partner. Piwi-interacting RNAs (piRNAs) bind Piwi-clade Argonaute proteins and act mainly in gonadal tissues to guard genome integrity by silencing mobile genetic elements (for review, see Malone and Hannon 2009).

Conceptually, the piRNA pathway can be divided into several different phases. During the initiation phase, small RNAs, called primary piRNAs, are produced from their generative loci, so-called piRNA clusters (Brennecke et al. 2007). These give rise to long, presumably single-stranded precursor transcripts, which are processed via an unknown biogenesis mechanism into small RNAs that are larger than canonical microRNAs (;24–30 nucleotides [nt]) (Aravin et al. 2006; Girard et al. 2006; Grivna et al. 2006;

Lau et al. 2006; Vagin et al. 2006). Primary piRNAs become stably associated with Piwi proteins to form Piwi RISCs (RNA-induced silencing complexes), which also contain additional proteins that facilitate target recognition and silencing. During the effector phase, Piwi RISCs identify targets via complementary base-pairing. In some cases, for example, with Aubergine as a piRNA partner, there is strong evidence for target cleavage in vivo (Brennecke et al. 2007; Gunawardane et al. 2007). This nucleolytic destruction of transposon mRNAs is probably the main Aubergine effector mechanism, although this has not been rigorously demonstrated. Piwi also conserves the Argo- naute catalytic triad; however, in this case, both its nuclear localization and its association with certain chromatin proteins suggest the possibility of transcriptional and post-transcriptional effector pathways (Brower-Toland et al. 2007; Klattenhoff et al. 2009; Saito et al. 2009). An additional phase, adaptation, is restricted to germ cells and constitutes the ping-pong cycle. During this phase, transposon mRNA cleavage directed by primary piRNAs triggers the production of secondary piRNAs, whose 59 ends correspond to cleavage sites (Brennecke et al. 2007; Gunawardane et al. 2007). These generally join Ago3 and enable it to recognize and cleave RNAs with antisense transposon content, perhaps piRNA cluster transcripts. Cleavage by Ago3 RISC again triggers piRNA production from the target, closing a loop that enables the overall small RNA population to adjust to challenge by a partic- ular transposon (for review, see Aravin et al. 2007). Finally, piRNA populations present in germ cells can be transmitted to the next generation to prime piRNA responses in progeny (Brennecke et al. 2008).

In Drosophila follicle cells, only the initiation and ef- fector phases appear relevant (Brennecke et al. 2008). Here, the piRNA pathway relies on the coupling between a single Piwi protein (Piwi itself) and a principal piRNA cluster (flamenco) to silence mainly gypsy family retro- transposons (Sarot et al. 2004; Saito et al. 2006; Brennecke et al. 2007; Li et al. 2009; Malone et al. 2009). Drosophila ovarian somatic sheet cells (OSS) display many of the properties of follicle cells, and represent a convenient system to study the initiation and effector phases of the piRNA pathway without the complications inherent in the study of complex tissues in vivo (Niki et al. 2006; Lau et al. 2009; Saito et al. 2009). We therefore sought to leverage information derived from the use of RNAi in OSS cells with the analysis of ovaries derived from mutant animals to probe the roles of known piRNA pathway components in the initiation and effector phases of trans- poson silencing.

Results and Discussion

The piRNA pathway is continuously required for transposon silencing

Several prior studies have proposed models in which Piwi proteins silence targets by interfering with their tran- scription (Pal-Bhadra et al. 2004). Since piRNAs are largely absent from somatic tissues (Cox et al. 2000), impacts underlying these changes are presumed to have occurred during development and to have been epigenetically maintained in the adult. Drosophila Piwi protein is mainly localized to the nucleus and has been shown to

[Keywords: Piwi; Zucchini; Armitage; Squash; primary piRNA; Dro- sophila] 3Corresponding author. E-MAIL [email protected]; FAX (516) 367-8874. Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1968110.

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interact with HP1, a core component of heterochromatin (Pal-Bhadra et al. 2004; Brower-Toland et al. 2007). Considered together, this body of evidence pointed strongly to an effector mechanism in which Piwi-associated small RNAs direct heterochromatin formation and silencing of targets.

Loss of piwi has dramatic effects on transposon expres- sion in somatic follicle cells (Sarot et al. 2004; Brennecke et al. 2007; Klenov et al. 2007; Malone et al. 2009). Genetic mutants result in an absence of Piwi protein throughout development. This could lead to a failure to create hetero- chromatic marks that could have otherwise maintained epigenetic silencing of transposons in the absence of continuous Piwi expression. Alternatively, there could be an ongoing requirement for Piwi to maintain silencing, irrespective of whether it acted via transcriptional or post-transcriptional mechanisms.

To discriminate between these possibilities, we trans- fected OSS cells with dsRNAs corresponding to piwi, and followed impacts on Piwi mRNA and protein levels (Supplemental Fig. S1A; data not shown). Maximal sup- pression was reached by 3 d, and silencing persisted through day 6. At 6 d post-transfection, we probed impacts on two elements known to be derepressed in the follicle cells of piwi mutant ovaries: gypsy and idefix. Both showed derepression (up to 10-fold) (Fig. 1A) upon piwi silencing. Additional elements were also tested (Supplemental Fig. S1B), with blood being impacted strongly. Previous studies have also implicated zucchini (zuc) in the function of the somatic piRNA pathway (Pane et al. 2007; Malone et al. 2009). RNAi against this gene also increased gypsy, blood, and idefix expression (Fig. 1A; Supplemental Fig. S1B). Considered together, these results demonstrate that the integrity of the piRNA pathway is essential for the ongoing repression of mobile elements and argue against a model in

which silent epigenetic states, once set by the action of piwi proteins on chromatin, can autonomously maintain transposon silencing.

Armitage is a component of the somatic piRNA pathway

Nearly a dozen proteins have been linked to the fully elaborated piRNA pathway that operates in germ cells (Malone et al. 2009). Many of these show germ cell-specific expression patterns consistent with their selective biolog- ical effects. Mutations in armitage (armi) result in co- incident loss of the characteristic nuclear accumulation of Piwi protein and a reduction in Piwi-associated piRNAs (Malone et al. 2009). Unlike most germline-specific path- way components, an examination of RNA-seq data from OSS cells indicated substantial armi expression (Supple- mental Fig. S1C). We therefore suppressed armi by RNAi and examined effects on transposon expression. Notably, gypsy, blood, and idefix were strongly derepressed, imply- ing a role for armi in both the somatic and germline compartments (Fig. 1A; Supplemental Fig. S1B).

The Drosophila mutant armi1 represents a P-element insertion in the 59 untranslated region (UTR) of armitage. A second allele, armi72.1, was derived from armi1 by imprecise excision (Cook et al. 2004). RNA-seq data covered the armi ORF in OSS, but no reads were detected corresponding to the germ cell 59 UTR (Supplemental Fig. S1D). This raises the possibility that armi expression might be driven by an alternative promoter in somatic cells, and that the armi alleles examined thus far may have spared the activity of that promoter.

Armitage and Zucchini function at the initiation phase

To investigate whether Armi and Zuc act at the initiation or effector phase of the piRNA pathway, we examined piRNAs. Silencing of piwi reduced levels of two abundant piRNAs, corresponding to gypsy, or idefix (Fig. 1B). Similar effects were noted upon silencing of armi or zuc. Aggregate OSS piRNA levels can be measured qualitatively by radioactive phosphate exchange of small RNAs in Piwi immunoprecipitates. As expected, RNAi against piwi virtually eliminated piRNAs in immunoprecipitates (Fig. 1C). Silencing of armi or zuc produced indistinguishable effects.

In germ cells, armi mutation causes loss of the prom- inent nuclear localization of Piwi (Malone et al. 2009). We observed a similar phenotype upon knockdown of armi in somatic OSS cells (Fig. 2A). Because of the mixed cell types present in ovaries, previous studies had not been able to distinguish whether Armi loss simply caused Piwi mislocalization or whether Armi influenced Piwi expres- sion or stability. In OSS cells, knockdown of armi reduced Piwi protein levels by approximately fivefold, equivalent to a targeted knockdown of Piwi itself without affecting piwi mRNA (Fig. 2B,C). We noted a similar loss of Piwi protein from the nuclei in cells exposed to zuc-dsRNAs (Fig. 2A). In this case, Piwi protein but not mRNA levels also fell (Fig. 2B,C).

Considered together, these data strongly suggest roles of Armi and Zuc in the initiation phase of the piRNA pathway. A role for Armi, along with a previously un- recognized component, Yb, in the somatic pathway, is also supported by a recent report from Brennecke and colleagues (Olivieri et al. 2010). Either protein could play

Figure 1. Effect of piwi, zucchini, and armitage knockdown on transposon silencing in OSS cells. OSS cells were treated with dsRNA against piwi, zuc, or armi for 6 d. qPCRs were normalized to internal controls rp49 (A) or bantam (B). Fold changes relative to cells treated with gfp-dsRNA are shown on a linear scale. Error bars represent one standard deviation over three biological replicates. Transcripts (A) and two abundant piRNAs (B) corresponding to gypsy and idefix retroelements were detected by qPCR. (C) Small RNAs coimmunoprecipitating with Piwi in untreated cells and cells treated with dsRNA against gfp, armi, zuci, or piwi were labeled with 32P at their 59 termini.

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a role in primary piRNA biogenesis, aiding piRNA pro- duction or loading, with this model resting on the pre- sumption that association with mature piRNAs influences Piwi protein stability (Fig. 3A, steps 1, 2). Alternatively, Armi or Zuc could be core components of mature Piwi RISC, with loss of either subunit destabilizing associated components of the complex (Fig 3A, step 3).

Armitage is a component of Piwi RISC

To investigate these alternative models, we performed proteomic analysis of Piwi RNPs. Piwi immunoprecipi- tates contained a number of peptides from Armi, suggest- ing that this protein is present in Piwi RISC (Fig. 3B). Of note, we also detected association of both Piwi and Armi with Squash (Squ), another previously identified piRNA pathway component (Pane et al. 2007). Piwi could be also detected in Squ immunoprecipitates by Western blotting. Although no Zuc peptides were seen in multidimensional protein identification technology (MudPIT), Piwi could be detected to a low extent in Zuc immunoprecipitates (Fig. 3C; Supplemental Fig. S2). Overall, the emerging picture suggests that both Armi and Squ are components of Piwi RISC. Lower levels of Piwi associated with Zuc might indicate a weaker or more transient association of Zuc with Piwi RISC.

Squash impacts the piRNA effector step

Mutations in squash (squ) show little impact on piRNA populations in mutant ovaries (Malone et al. 2009). Simi- larly, upon sequencing of small RNAs in Piwi immunopre- cipitates, we failed to detect any differences in associated piRNA populations upon comparison of squ homozygous mutant animals to heterozygous siblings (Fig. 4B). Animals harboring two squ alleles interrupted by early stop codons did, however, display an effect on transposon silencing.

As compared with heterozygous siblings, squ mutants showed significant derepression of gypsy (Fig. 5A). This occurred without any detectable change in an abundant gypsy piRNA or overall Piwi levels (Fig. 5B,C). In con- trast, no substantial changes were detected in idefix or

ZAM (Fig. 5A,B); however, I-element and blood were strongly derepressed (Supple- mental Fig. S3).

Considered together, these results point to a role of squash in the effector phase of the piRNA pathway. We did note a slight but reproducible reduction in Piwi protein levels in homozygous squ mu- tants (Fig. 5C; Supplemental Fig. S4). However, this was well within the range observed in Piwi heterozygotes, where the piRNA pathway functions completely normally.

A possible role for Zucchini in piRNA biogenesis

In the initial screen that placed zuc within the piRNA pathway, two alleles were identified (Schupbach and Wieschaus 1991; Pane et al. 2007). zucHM27 represents an early stop mutation resulting in a puta- tive null allele (referred to as zuc mut). This mutant strongly affects piRNA si-

lencing in both germline and somatic cells of the ovary (Pane et al. 2007; Malone et al. 2009). While somatic piRNAs are depleted in this mutant, ping-pong signatures remain intact (Malone et al. 2009). This places Zuc outside of the adaptive phase, consistent with our accumulating evidence for a role in the initiation phase.

While the biochemical properties of Zuc have yet to be analyzed, its protein sequence places it as a member of the phospholipase D (PLD) family of phosphodiester- ases. These share a HxK(x)4D motif, whose integrity is

Figure 2. Piwi protein localization and levels upon knockdown of armitage or zucchini. (A) Piwi subcellular localization was determined by immunostaining in OSS cells treated with zuc-dsRNA, armi-dsRNA, or piwi-dsRNA. Gfp-dsRNA-treated cells were used as control. (B) Piwi protein levels in total cell extracts were determined by Western blotting. Tubulin was used as a loading control. (D) Piwi qPCRs were normalized to rp49. Fold changes relative to cell treated with gfp-dsRNA are shown on a linear scale. Error bars represent one standard deviation over three biological replicates.

Figure 3. MudPIT analysis of Piwi, Zucchini, and Squash com- plexes. (A) A proposed model for the function of piRNA pathway components is shown. (B) Protein associations identified by MudPIT are depicted. Arrows point from the immunoprecipitated protein to the coimmunoprecipitated protein. Numbers of identified peptides and corresponding unique sequences (shown in parentheses) of two biological replicates are indicated. Only peptides above the signifi- cance threshold were considered (see the Materials and Methods). (C) Zucchini, Squash, and Piwi were immunoprecipitated (IP) from OSS cells. The presence of Piwi was assessed by Western blotting.

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essential for catalytic activity (Zhao et al. 1997; Sasnauskas et al. 2010). The second zuc mutation that emerged in the original screen, zucSG63, contains a H / Y mutation within the phosphodiesterase motif that is predicted to render it catalytically inactive. To probe a role for Zuc catalytic activity in the piRNA pathway, we compared the presumed null (zuc mut) and catalytically dead (zuc H / Y) alleles for their effects on piRNAs and transposon silencing.

We analyzed total ovarian small RNAs from animals that were heterozygous or homozygous for the zuc H / Y allele and compared the resulting profiles to previously published analyses of the presumed zuc mut allele (Malone et al. 2009). In both cases, we saw strong re- ductions in total piRNAs and in populations that mapped uniquely to the flamenco locus (Fig. 4A), regardless of the normalization method used to compare libraries (Supple- mental Fig. S5). Slightly stronger impacts were apparent when we compared profiles of Piwi immunoprecipitates (Fig. 4B). Here, piRNA populations corresponding to fla- menco were almost completely lost. We did note an accumulation of 21-nt species in Piwi immunoprecipi- tates from both zuc mutant lines. These were enriched

for a 59 U, although not to the extent for longer piRNA species. The nature of these shorter, apparently Piwi- associated RNAs remains mysterious.

Both the presumed null and H / Y zuc alleles impacted transposon silencing (Fig. 5A). Between fivefold and 20-fold increases in gypsy, ZAM, and idefix were noted in comparison with heterozygous controls. Even stronger derepression could be observed for I-element, HeT-A, 1731, and blood (Supplemental Fig. S3). The zuc H / Y and zuc mut alleles also showed similar impacts on piRNA populations (Fig. 5B) and the overall levels of Piwi protein (Fig. 5C; Supplemental Fig. S4).

Considered together, these data point to a requirement for the presumed catalytic center of Zuc in the initiation phase of the piRNA pathway. Other PLD family nu- cleases that have been characterized to date cleave nu- cleic acids leaving 59 phosphate and 39 hydroxyl termini (Pohlman et al. 1993; Zhao et al. 1997; Sasnauskas et al. 2010). These are the characteristics one might expect for a processing enzyme that catalyzed primary piRNA bio- genesis. Previous studies have posited the requirement for several nucleolytic activities in the piRNA pathway. One is thought to form the 59 ends of primary piRNAs. The

Figure 4. piRNA populations from zucchini and squash mutant ovaries. Heterozygous siblings serve as control. (Left panel) Size profiles of small RNAs mapping to repeats normalized to total read counts are shown. (Right panel) Densities of uniquely mapping piRNAs are plotted over the flamenco locus in reads per million (only small RNAs matching the plus strand are depicted). (A) Small RNAs cloned from total RNA. (B) Small RNAs from Piwi immunoprecipitates (IP). Regions of flamenco measured in Figure 5D are indicated by asterisks.

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39 ends of these species could be formed prior to Piwi loading or could be coupled to protein binding, as is posited for the ping-pong cycle. The nucleolytic center of Piwi proteins themselves form the 59 ends of secondary piRNAs, with their 39 ends proposed to be created by a separate enzyme. Based on its impacts in the soma on Piwi complexes, we imagined that the Zuc catalytic center might form either the 59 or 39 ends of primary piRNAs.

To evaluate this hypothesis, we examined RNAs de- rived from the flamenco locus in control ovaries or in tissues from animals homozygous for either of the two zuc mutant alleles. The prevailing model holds that the flamenco locus is transcribed as a continuous, single- stranded precursor spanning >150 kb (Brennecke et al. 2007). We reasoned that a defect in primary processing might result in an accumulation of long RNAs from this locus, since they would not be effectively metabolized into piRNAs. By quantitative PCR (qPCR) using primer pairs spanning three different regions of flamenco (see Fig. 4B, right panel), we saw 15-fold to 45-fold increases in flamenco-derived long RNAs in zuc mutant ovaries (Fig. 5D; Supplemental Fig. S6).

Considered as a whole, our results strongly support a role for Zucchini in the primary processing of piRNAs from the flamenco locus. Given its size, it is virtually impossible to follow the fate of the intact flamenco transcript by Northern blotting. Three different segments of the locus do show an accumulation consistent with

their failure to be parsed into piRNAs. However, several alternative explanations can also be envisioned. For exam- ple, if Zucchini impacts Piwi stability, feedback controls might operate to inhibit primary biogenesis. Without a direct, biochemical demonstration that Zucchini pro- cesses piRNA cluster transcripts, its assignment as a primary biogenesis enzyme must be viewed as provi- sional. However, any alternative model must account for the requirement for its phosphodiesterase active site, and, at present, a direct role in piRNA biogenesis seems the most parsimonious conclusion.

While our studies do not ascribe specific functions to Armitage and Squash, they do support their assignment to the initiator and effector phases, respectively. Armitage is a putative helicase, although no analyses as yet indicate whether this biochemical activity is required for its function. Our placement of this protein in the initiation phase and its intimate association with Piwi perhaps suggest a role in loading or stability of Piwi RISC. Squash, of all the components examined in this study, had the most variable effects on transposon control in somatic cells of the ovary (Figs. 1A, 5A), but both its physical association with Piwi RISC and its impact on transposons without an effect on piRNAs imply a role in the effector phase. While the studies reported herein can suggest roles for known pathway components at specific points in the piRNA pathway, a definitive conclusion regarding the part played by any of these proteins will require recon- stitution of the pathway in vitro.

Materials and methods

OSS cell culture and knockdown

The OSS cell line was a kind gift from Yuzo Niki, and was cultured as

described (Niki et al. 2006). dsRNA was prepared as described (http://

www.flyrnai.org/DRSC-PRS.html). Cells were transfected with dsRNA

using Xfect Transfection Reagent (Clontech). Six days after transfection,

RNA and protein were extracted. Detailed information for RT-qPCR,

Western blotting, and immunofluorescence is available in the Supple-

mental Material.

Fly stocks and allelic combinations

Fly stocks and allelic combinations used were squ mut: squHE47/PP32

(Pane et al. 2007); zuc mut: zucHM27/Df(2I)PRL (Pane et al. 2007); and zuc

H / Y: zucHM27/SG63 (Pane et al. 2007).

Preparation of small RNA libraries

Small RNA libraries were prepared as described (Brennecke et al. 2007).

Detailed information is available in the Supplemental Material.

Acknowledgments

We thank Vasily Vagin, Anna Jankowska, Adam Rosebrock, Yaniv Erlich,

Julius Brennecke, Alexei Aravin, Delphine Fagegaltier, and current and

former members of the Hannon laboratory for helpful discussions and

support. The OSS cell line was a gift of Yuzo Niki (Ibaraki University). Fly

stocks were kindly provided by Gertrud Schupbach (Princeton). PMG is

supported by Grant 5T32GM065094 from the NIH and by a William

Randolph Hearst Foundation Scholarship. S.F. is supported by the Elizabeth

Sloan Livingston Foundation. B.C. is supported by the Boehringer Ingelheim

Fonds. G.J.H. is an investigator of the Howard Hughes Medical Institute.

This work was supported by grants from the NIH (to G.J.H.). Sequences

reported in this study can be found at GEO using accession number

GSE24108 (which includes libraries GSM593297–593304).

Figure 5. Transposons, piRNAs, and piRNA precursors in zucchini and squash mutant ovaries. (A) Transcripts of gypsy, idefix, and ZAM transposons were detected by qPCR. (B) Individual piRNAs targeting gypsy and idefix were detected by qPCR. (C) Piwi protein levels in mutant and heterozygous ovary extracts were measured by Western blotting. Tubulin serves as loading control. Celera sequenc- ing strain (S-strain) is shown in addition. (D) Three ;100-nt regions of flamenco that are normally highly processed into piRNAs were detected by qPCR. The positions of these segments are indicated in Figure 4. qPCR data were normalized to internal controls rp49 (A,C) or bantam (B). Fold changes relative to heterozygous siblings are shown on a linear scale. Error bars represent one standard deviation over three technical replicates.

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References

Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N,

Morris P, Brownstein MJ, Kuramochi-Miyagawa S, Nakano T, et al.

2006. A novel class of small RNAs bind to MILI protein in mouse

testes. Nature 442: 203–207.

Aravin AA, Hannon GJ, Brennecke J. 2007. The Piwi–piRNA pathway

provides an adaptive defense in the transposon arms race. Science

318: 761–764.

Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R,

Hannon GJ. 2007. Discrete small RNA-generating loci as master

regulators of transposon activity in Drosophila. Cell 128: 1089–

1103.

Brennecke J, Malone CD, Aravin AA, Sachidanandam R, Stark A, Hannon

GJ. 2008. An epigenetic role for maternally inherited piRNAs in

transposon silencing. Science 322: 1387–1392.

Brower-Toland B, Findley SD, Jiang L, Liu L, Yin H, Dus M, Zhou P, Elgin

SC, Lin H. 2007. Drosophila PIWI associates with chromatin and

interacts directly with HP1a. Genes Dev 21: 2300–2311.

Cook HA, Koppetsch BS, Wu J, Theurkauf WE. 2004. The Drosophila

SDE3 homolog armitage is required for oskar mRNA silencing and

embryonic axis specification. Cell 116: 817–829.

Cox DN, Chao A, Lin H. 2000. piwi encodes a nucleoplasmic factor

whose activity modulates the number and division rate of germline

stem cells. Development 127: 503–514.

Ghildiyal M, Zamore PD. 2009. Small silencing RNAs: An expanding

universe. Nat Rev Genet 10: 94–108.

Girard A, Sachidanandam R, Hannon GJ, Carmell MA. 2006. A germline-

specific class of small RNAs binds mammalian Piwi proteins. Nature

442: 199–202.

Grivna ST, Beyret E, Wang Z, Lin H. 2006. A novel class of small RNAs in

mouse spermatogenic cells. Genes Dev 20: 1709–1714.

Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y, Nagami

T, Siomi H, Siomi MC. 2007. A slicer-mediated mechanism for

repeat-associated siRNA 59 end formation in Drosophila. Science

315: 1587–1590.

Klattenhoff C, Xi H, Li C, Lee S, Xu J, Khurana JS, Zhang F, Schultz N,

Koppetsch BS, Nowosielska A, et al. 2009. The Drosophila HP1

homolog Rhino is required for transposon silencing and piRNA

production by dual-strand clusters. Cell 138: 1137–1149.

Klenov MS, Lavrov SA, Stolyarenko AD, Ryazansky SS, Aravin AA,

Tuschl T, Gvozdev VA. 2007. Repeat-associated siRNAs cause chro-

matin silencing of retrotransposons in the Drosophila melanogaster

germline. Nucleic Acids Res 35: 5430–5438.

Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, Bartel DP,

Kingston RE. 2006. Characterization of the piRNA complex from rat

testes. Science 313: 363–367.

Lau NC, Robine N, Martin R, Chung WJ, Niki Y, Berezikov E, Lai EC.

2009. Abundant primary piRNAs, endo-siRNAs, and microRNAs in

a Drosophila ovary cell line. Genome Res 19: 1776–1785.

Li C, Vagin VV, Lee S, Xu J, Ma S, Xi H, Seitz H, Horwich MD, Syrzycka

M, Honda BM, et al. 2009. Collapse of germline piRNAs in the

absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137:

509–521.

Malone CD, Hannon GJ. 2009. Small RNAs as guardians of the genome.

Cell 136: 656–668.

Malone CD, Brennecke J, Dus M, Stark A, McCombie WR, Sachidanandam

R, Hannon GJ. 2009. Specialized piRNA pathways act in germline and

somatic tissues of the Drosophila ovary. Cell 137: 522–535.

Niki Y, Yamaguchi T, Mahowald AP. 2006. Establishment of stable cell

lines of Drosophila germ-line stem cells. Proc Natl Acad Sci 103:

16325–16330.

Olivieri D, Sykora M, Sachidanandam R, Mechtler K, Brennecke J. 2010.

An in vivo RNAi assay identifies major genetic and cellular re-

quirements for primary piRNA biogenesis in Drosophila. EMBO

J doi: 10.1038/emboj.2010.212.

Pal-Bhadra M, Leibovitch BA, Gandhi SG, Rao M, Bhadra U, Birchler JA,

Elgin SC. 2004. Heterochromatic silencing and HP1 localization in

Drosophila are dependent on the RNAi machinery. Science 303: 669–

672.

Pane A, Wehr K, Schupbach T. 2007. zucchini and squash encode two

putative nucleases required for rasiRNA production in the Drosophila

germline. Dev Cell 12: 851–862.

Pohlman RF, Liu F, Wang L, More MI, Winans SC. 1993. Genetic and

biochemical analysis of an endonuclease encoded by the IncN

plasmid pKM101. Nucleic Acids Res 21: 4867–4872.

Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, Siomi

H, Siomi MC. 2006. Specific association of Piwi with rasiRNAs

derived from retrotransposon and heterochromatic regions in the

Drosophila genome. Genes Dev 20: 2214–2222.

Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, Sakota E, Kotani H,

Asai K, Siomi H, Siomi MC. 2009. A regulatory circuit for piwi by the

large Maf gene traffic jam in Drosophila. Nature 461: 1296–1299.

Sarot E, Payen-Groschene G, Bucheton A, Pelisson A. 2004. Evidence for

a piwi-dependent RNA silencing of the gypsy endogenous retrovirus

by the Drosophila melanogaster flamenco gene. Genetics 166: 1313–

1321.

Sasnauskas G, Zakrys L, Zaremba M, Cosstick R, Gaynor JW, Halford SE,

Siksnys V. 2010. A novel mechanism for the scission of double-

stranded DNA: BfiI cuts both 39–59 and 59–39 strands by rotating

a single active site. Nucleic Acids Res 38: 2399–2410.

Schupbach T, Wieschaus E. 1991. Female sterile mutations on the second

chromosome of Drosophila melanogaster. II. Mutations blocking

oogenesis or altering egg morphology. Genetics 129: 1119–1136.

Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD. 2006. A

distinct small RNA pathway silences selfish genetic elements in the

germline. Science 313: 320–324.

Zhao Y, Stuckey JA, Lohse DL, Dixon JE. 1997. Expression, characteriza-

tion, and crystallization of a member of the novel phospholipase

D family of phosphodiesterases. Protein Sci 6: 2655–2658.

Haase et al.

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