The Bombyx Ovary-Derived Cell Line Endogenously Expresses PIWI/PIWI-Interacting RNA Complexes

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The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes

SHINPEI KAWAOKA,1 NOBUMITSU HAYASHI,1 YUTAKA SUZUKI,2 HIROAKI ABE,3 SUMIO SUGANO,2 YUKIHIDE TOMARI,4,5,6 TORU SHIMADA,1 and SUSUMU KATSUMA1 1Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan 2Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639, Japan 3Division of Agriscience and Bioscience, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan 4Institute of Molecular and Cellular Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 113-0032, Japan 5Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 113-0032, Japan 6PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

ABSTRACT Genetic studies and large-scale sequencing experiments have revealed that the PIWI subfamily proteins and PIWI-interacting RNAs (piRNAs) play an important role in germ line development and transposon control. Biochemical studies in vitro have greatly contributed to the understanding of small interfering RNA (siRNA) and microRNA (miRNA) pathways. However, in vitro analyses of the piRNA pathway have been thus far quite challenging, because their expression is largely restricted to the germ line. Here we report that Bombyx mori ovary-derived cultured cell line, BmN4, endogenously expresses two PIWI subfamily proteins, silkworm Piwi (Siwi) and Ago3 (BmAgo3), and piRNAs associated with them. Siwi-bound piRNAs have a strong bias for uridine at their 59 end and BmAgo3-bound piRNAs are enriched for adenine at position 10. In addition, Siwi preferentially binds antisense piRNAs, whereas BmAgo3 binds sense piRNAs. Moreover, we identified many pairs in which Siwi-bound antisense and BmAgo3-bound sense piRNAs are overlapped by precisely 10 nt at their 59 ends. These signatures are known to be important for secondary piRNA biogenesis in other organisms. Taken together, BmN4 is a unique cell line in which both primary and secondary steps of piRNA biogenesis pathways are active. This cell line would provide useful tools for analysis of piRNA biogenesis and function. Keywords: PIWI; piRNA; cultured cell; Bombyx mori; BmN4

INTRODUCTION quencing efforts in several organisms including mammals have identified the presence of piRNAs matching trans- PIWI subfamily proteins bind to a class of small RNAs, posons (O’Donell and Boeke 2007). Thus, PIWI proteins called PIWI-interacting RNAs (piRNAs). Insights into and piRNAs are thought to exert an essential role in germ PIWI and piRNAs have been obtained mainly from genetic line development, stem cell self-renewal, and transposon studies and large-scale sequencing. Genetic studies have control (Hartig et al. 2007; O’Donell and Boeke 2007; implicated PIWI proteins in germ line development in Klattenhoff and Theurkauf 2008). Unlike small interfering both invertebrates and vertebrates. In Drosophila, piwi mu- RNAs (siRNAs) and microRNAs (miRNAs), piRNAs are tations cause sterility and loss of germ line stem cells. The produced by a Dicer-independent mechanism (Vagin et al. requirement of piwi for transposon control has been also 2006; Houwing et al. 2007). Brennecke et al. (2007) and shown genetically in Drosophila and mouse (Hartig et al. Gunawardane et al. (2007) proposed a ping-pong model of 2007; Klattenhoff and Theurkauf 2008). Large-scale se- piRNA production. D. melanogaster encode three PIWI subfamily proteins; Piwi, Aubergine (Aub), and Argo- Reprint requests to: Susumu Katsuma, Department of Agricultural and naute3 (Ago3) (Brennecke et al. 2007; Gunawardane et al. Environmental Biology, Graduate School of Agricultural and Life Sciences, 2007). In the ping-pong model, Piwi/Aub-bound antisense University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan; piRNAs direct sense piRNA cleavage, resulting in the e-mail: [email protected]; fax: 81-3-5841-8993. Article published online ahead of print. Article and publication date are production of Ago3-bound sense piRNAs, and vice versa at http://www.rnajournal.org/cgi/doi/10.1261/rna.1452209. (Brennecke et al. 2007; Gunawardane et al. 2007).

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Kawaoka et al.

Biochemical analysis using lysates from Drosophila embryos and S2 cells has provided many valuable insights into the biogenesis and functions of siRNAs and miRNAs (Tuschl et al. 1999; Zamore et al. 2000; Bernstein et al. 2001; Elbashir et al. 2001; Hutva´gner et al. 2001; Zamore 2001). However, piRNA expression is largely restricted to the germ line, which makes it difficult to obtain enough lysate for in vitro experiments. Here we report that the silkworm cell line BmN4 endogenously expresses two PIWI proteins and piRNAs associated with them. This cell line can be used as a robust model system for monitoring the piRNA-associated pathway.

RESULTS AND DISCUSSION

Identification and characterization of piRNAs in BmN4 cells Based on our previous observations of a huge amount of piRNAs in the silkworm Bombyx mori ovary (Kawaoka et al. 2008b), we sought for piRNA-like small RNA-expressing cell lines derived from ovaries of several lepidopteran insect species to establish an in vitro ex- perimental system for piRNA research. FIGURE 1. Identification and characterization of BmN4-derived small RNAs. (A) Total RNA As shown in Figure 1A, we found that (10 mg) from three lepidopteran cultured cell lines (BmN4, High Five, and Sf9) were loaded on a distinct population of small RNAs a 15% polyacrylamide gel containing 10 M urea, electrophoresed, and stained with SYBRGold. Molecular sizes are shown to the left of the figure. The arrow indicates the small RNA species of around 27–28 nucleotides (nt) long are interest. (B) Length distributions of sequenced BmN4-derived piRNAs. BmN4-derived piRNAs expressed in Bombyx-derived BmN4, displayed a unimodal length distribution with a peak at 27 nt. (C) Nucleotide compositions of Trichoplusia ni-derived High Five, and sequenced total BmN4-derived piRNAs. BmN4-derived piRNAs have a strong bias toward Spodoptera frugiperda-derived Sf9 cells. uridine at the 59 end (76%). (D) Pie chart summarizing the annotation of BmN4-derived small RNAs. (E) Chemical characterization of 39 end of Bombyx piRNA. Northern blot analysis of In this study, we focused on BmN4- ovary- and BmN4-derived total RNAs after NaIO4-mediated oxidation followed by derived small RNAs, as the Bombyx b-elimination reaction (indicated by +) was performed. Molecular sizes are shown to the genome has been almost completely se- left of the figure and DNA probes used are indicated above. quenced (The International Silkworm Genome Consortium 2008). Solexa sequencing technology was used to generate 416,177 species from BmN4 cells and Bombyx ovary. In the case of SART1, (1,058,466 clones) of unique small RNAs with 0–2 mis- ovary-derived small RNAs were mapped almost exclusively matches in the Bombyx genome. Seventy-five percent of to ORF2 (Kawaoka et al. 2008b), while this feature was not cloned small RNAs were read only once, suggesting that observed in BmN4-derived small RNAs (Supplemental Fig. they are highly diverse. Cloned small RNAs showed a 1). In addition, the number of BmN4-derived small RNAs Gaussian distribution in size with a peak at 27 nt (Fig. matching HOPEBm2 was much less than that of ovary- 1B). Of these, 76% contained uridine (U) at the 59 end (Fig. derived small RNAs (Supplemental Fig. 1). These differ- 1C), a bias similar to that observed in piRNAs from other ences might be due to different activities or copy numbers organisms. Classification of cloned small RNAs in Figure of transposons in each source. 1D shows that 31% of them were associated with repetitive Previous studies have shown that the 39 ends of piRNAs sequences. Relationships between transposons and BmN4- are 29-O-methylated (Horwich et al. 2007; Kirino and derived small RNAs were indicated in Supplemental Table Mourelatos 2007; Ohara et al. 2007; Saito et al. 2007). To 1. We observed differences in small RNA profiles derived investigate properties of the 39 ends of BmN4-derived small

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Bombyx cultured cells as a model for piRNA study

RNAs, we performed NaIO4-mediated oxidation followed antibody did not detect any bands from the anti-FLAG by b-elimination reaction to ovary- and BmN4-derived immunoprecipitates from extracts of BmN4 cells stably total RNAs. As shown in Figure 1E, Northern blot analysis expressing FLAG-BmAgo3 or FLAG-Siwi, respectively. of piRNA-1, one of the abundant small RNAs in BmN4 Therefore, we concluded that anti-Siwi and anti-BmAgo3 cells, and Bombyx let-7 revealed that these reactions did not antibodies could specifically recognize Siwi and BmAgo3, affect the mobility of piRNA-1, while the reaction product respectively, and both proteins were not coimmunopreci- of let-7 migrated faster than that without reaction (Fig. 1E), pitated. Collectively, piRNAs and PIWI subfamily proteins indicating that the 39-end of small RNAs cloned from exist in BmN4 cells. BmN4 cells is blocked. Together with these results, we refer to cloned small RNA as piRNA in the following sections. Evidence for piRNA biogenesis in BmN4 cells To characterize the piRNA population in each PIWI Expression of two Bombyx PIWI proteins, Siwi complex in BmN4 cells, we immunopurified FLAG-Siwi- and BmAgo3, in BmN4 cells or FLAG-BmAgo3-containing complexes from cells stably Next, we investigated whether PIWI proteins exist in BmN4 expressing FLAG-Siwi or FLAG-BmAgo3, respectively, using cells. In Bombyx, the PIWI subfamily consists of two anti-FLAG antibody, as antibodies against endogenous Siwi members, Siwi and BmAgo3 (Kawaoka et al. 2008a). To and BmAgo3 were not suitable for coimmunoprecipitation examine the endogenous expression of Siwi and BmAgo3, of PIWI-bound piRNAs (data not shown). Subsequently, we generated rabbit polyclonal antibody against each small RNAs in each complex were cloned and deeply protein. Western blot analysis using anti-Siwi and sequenced. We obtained 6,034,838 and 4,398,344 clones of anti-BmAgo3 antibodies revealed that endogenous Siwi Siwi- and BmAgo3-bound piRNAs, respectively. Siwi-bound and BmAgo3 were expressed as z100 kDa and 110 kDa piRNAs showed a strong enrichment for U at position 1 proteins in BmN4 cells, respectively (Fig. 2). In addition, (77%), while BmAgo3-bound piRNAs lacked this bias cell lines stably expressing FLAG-tagged Siwi or FLAG- (Fig. 3A). In contrast, position 10 of BmAgo3-bound tagged BmAgo3 were generated. The specificities of anti- piRNAs was enriched for A (75%) (Fig. 3B). These biases Siwi and anti-BmAgo3 antibodies were verified using these are quite similar to those observed in Drosophila PIWI cell lines (Supplemental Fig. S2). Anti-Siwi or anti-BmAgo3 subfamily-bound piRNAs (Brennecke et al. 2007). Next, to investigate relationships between Siwi- or BmAgo3-bound piRNAs and transposons, we focused on piRNAs that matched 120 transposons (Fig. 4A; Supple- mental Tables 2, 3). We observed a clear trend toward antisense and sense preferences in Siwi- and BmAgo3-bound piRNAs, respectively (Fig. 4A,B). Density maps of piRNAs on Aquila and SART1 transposons clearly showed that antisense piRNAs are preferentially incorporated into Siwi- containing complexes (Fig. 4C, red line), while sense piRNAs are preferentially incorporated into BmAgo3-containing complexes (Fig. 4C, blue line). In addition, as reported in zebrafish (Houwing et al. 2008), we observed an opposite piRNA polarity in some transposons (Fig. 4A,C). Sense–antisense piRNA pairs overlapping by precisely 10 nt at their 59 ends are characteristic of secondary piRNA biogenesis in the ping-pong model (Brennecke et al. 2007; Gunawardane et al. 2007). To verify whether secondary FIGURE 2. Expressions of Siwi and BmAgo3 in BmN4 cells. (A) piRNA biogenesis pathway exists in BmN4 cells, we focused Expression of Siwi in BmN4 cells. Western blotting was performed using total protein extract from BmN4 cells. The immunoprecipitates on the complementary relationship between Siwi-bound prepared with anti-FLAG antibody from extracts of BmN4 cells stably piRNAs and BmAgo3-bound piRNAs. As shown in Figure expressing FLAG-Siwi were used as a positive control. Siwi was 5A, many Siwi-bound antisense and BmAgo3-bound sense z expressed as an 100-kDa protein in BmN4 cells. (B) Expression of piRNA pairs are overlapped by precisely 10 nt from their 59 BmAgo3 in BmN4 cells. BmAgo3 was not detected by anti-BmAgo3 antibody using total protein lysate from BmN4 cells (data not shown). ends. Also, we found the strongest complementarity Thus, Western blotting was performed using the immunoprecipitates between piRNAs in Siwi and BmAgo3 (Fig. 5B). 59 10-nt prepared with anti-BmAgo3 antibody from extracts of BmN4 cells. overlapping was also observed both within Siwi-bound and The immunoprecipitates prepared with anti-FLAG antibody from BmAgo3-bound piRNAs. Such self-complementarity has extracts of BmN4 cells stably expressing FLAG-BmAgo3 were used as a positive control. BmAgo3 was expressed as an z110 kDa protein in been observed in Drosophila ovary-derived piRNAs (Bren- BmN4 cells. necke et al. 2007). A recent report suggests that piRNAs

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Kawaoka et al.

cultured at 27°C in TC-100 medium (Invitrogen) supplemented with 10% fetal bovine serum and High Five cells were cultured at 27°C in Express Five medium (Invitrogen), respectively.

RNA isolation Total RNAs were prepared using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. For small RNA preparation, miRVana miRNA isolation kit (Ambion) was used according to the manufacturer’s protocol.

Detection of small RNAs and preparation of small RNA libraries Total RNA (10 mg) was loaded onto a 15% denaturing polyacrylamide gel containing 10 M urea, electrophoresed, and then stained with SYBRGold (Invitrogen). Signals were visualized using LAS-1000 film (Fujifilm). Small RNA libraries were constructed using a Small RNA cloning kit (Takara). DNA sequencing was performed using the Solexa genetic analysis system (Illumina) (Bently 2006). A total of 1 ng of the prepared cDNA was used for the sequencing reactions with the Illumina GA; 10,000–15,000 clusters were generated per ‘‘tile’’ and 36 cycles of the sequencing reactions were performed. The protocols of the cluster generation and sequence reactions were according to the manufacturer’s FIGURE 3. Nucleotide biases of Siwi- and BmAgo3-bound piRNAs instructions in BmN4 cells. (A) Nucleotide bias of Siwi-bound piRNAs. (B) Nucleotide bias of BmAgo3-bound piRNAs. Sequence analysis of cloned small RNAs Solexa sequencing generated reads of up to 36 nt in length. The 39 which have U at position 1 but do not have A at position 10 adaptor sequences were identified and removed, allowing for up (1U but not 10A) are putative primary piRNAs, and those to two mismatches. Reads without adaptor sequences were showing 10A but not 1U are secondary piRNAs (Aravin discarded. Reads shorter than 23 nt or longer than 30 nt were et al. 2008). Strikingly, we found that z80% of Siwi-bound excluded, resulting in reads of 23–30 nt. The trimming described antisense piRNAs that contain 1U (but not 10A) form 10 nt above and alignment to the Bombyx genome (The International overlapping pairs with BmAgo3-bound sense piRNAs Silkworm Genome Consortium 2008) were performed using having 10A (but not 1U). These results strongly indicated SOAPaligner/soap2 (Li et al. 2008). Reads that could be aligned that both primary and secondary steps of piRNA biogenesis to the genome up to two mismatches were used for further pathways are active in BmN4 cells. analysis. Alignments of the extracted reads onto the annotated transposable elements in the Bombyx genome were performed using BLAST, allowing no mismatch. CONCLUSION b-Elimination Here we identified PIWI proteins and piRNAs endogenously expressed in the Bombyx-derived cell line, BmN4. Our data NaIO4 reaction was performed by incubating total RNA in 25 show that both of the primary and secondary piRNA mM NaIO4 at room temperature for 30 min. Ten microliters of biogenesis pathways are active in BmN4 cells. This is the glycerol were added to quench unreacted NaIO4. b-Elimination was then performed by adding 10 mL of 500 mM NaOH followed first discovery of the cultured cell line expressing the PIWI/ by incubation at 45°C for 90 min. The resultant RNA was piRNA complexes. Just as cultured cell lines including collected by ethanol precipitation. Drosophila S2 and human HeLa cells have largely contributed to progress in the study on siRNAs and miRNAs, Northern blot BmN4 cells would provide extremely valuable tools for biochemical understanding of the piRNA pathway. Total RNA was separated on urea-containing 15% polyacrylamide gel and transferred to Hybond-N+ (GE Healthcare Biosciences) using the Trans-Blot SD semidry electrophoretic transfer cell (Bio- MATERIALS AND METHODS Rad). For the detection of piRNA-1 and Bombyx let-7, DNA probes (59-ATTCGAAACCAATCCGTTAGTTTTTGA-39 for Cell lines piRNA-1 and 59-TACTATACAACCTACTACCTCA-39 for let-7) were radiolabeled with T4 polynucleotide kinase (Takara Bio Inc.) BmN4 cells were cultured at 27°C in IPL-41 medium (Gibco) and [g-32P] ATP. The probes were hybridized using Perfecthyb supplemented with 10% fetal bovine serum. The Sf9 cells were Plus (Sigma) at 37°C.

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Kawaoka et al.

FIGURE 5. The 10-nt overlap from 59 ends between Siwi- and BmAgo3-bound piRNAs. (A) The 10- nt overlap between Siwi-bound antisense piRNAs and BmAgo3-bound sense piRNAs. We calculated the number of ping-pong ‘‘sites’’ within each transposons and plotted total numbers of sites at each overlapping length. The number of overlapping sites is shown along the y-axis. The overlapping length at the 59 end is indicated along the x-axis. (B) Shown are heat maps that indicate the frequency to which 10-nt overlapping sense–antisense piRNA pairs from position 1–10 are found. Total small RNAs, SIWI-bound piRNAs, and BmAgo3-bound piRNAs were used in this analysis. Right panel indicates a control analysis performed with the 9-nt overlap.

Antibody generation FLAG-tagged Siwi and BmAgo3 was verified using the previously described Western blot analysis (Katsuma et al. 2005) with anti- Amplification products corresponding to amino acids 1–200 of FLAG monoclonal (1:1000, Sigma), anti-Siwi (1:10,000), or anti- Siwi and amino acids 80–230 of BmAgo3 were cloned into the BmAgo3 (1:5000) antibodies. pET24b vector (Novagen) and expressed in Escherichia coli BL21 DE3 at 37°C in the presence of 1 mM IPTG. Recombinant Immunoprecipitation of Siwi-bound proteins were purified using a HisGraviTrap column (GE Health- and BmAgo3-bound piRNAs care Bioscience) according to the manufacturer’s protocol. The purified recombinant proteins were used to generate anti-Siwi and We used anti-FLAG antibody to immunoprecipitate Siwi and anti-BmAgo3 rabbit polyclonal antibodies (Sigma). BmAgo3 from cells stably expressing FLAG-Siwi or FLAG- BmAgo3. The cells were cultured in a 150-mm dish and collected Generation of BmN4 cells stably expressing with RIPA buffer (50 mM Tris at pH 8.0, 100 mM NaCl, 3 mM FLAG/His-tagged PIWI proteins MgCl2, 1% NP-40, protease inhibitor cocktail (Roche), 100 U/mL RNasin ribonuclease inhibitor (Promega). The lysate was cleared BmN4 cells stably expressing FLAG/His-tagged Siwi or FLAG/His- by centrifugation at 15,000 rpm for 15 min at 4°C. The total tagged BmAgo3 were generated as described previously (Katsuma protein concentration was determined using Coomassie Plus et al. 2006). The full-length cDNA of Siwi was cloned into pIZ/V5- Protein assay reagent (Pierce) according to the manufacturer’s His (Invitrogen) and PCR-mediated mutagenesis was performed protocol. The resulting supernatant was collected and incubated to attach an N-terminal FLAG/His-tagged sequence (primers are with anti-FLAG antibody (1:100) or normal mouse IgG (Santa indicated in Supplemental Table 4). The coding region of Cruz; 1:40) for 2 h with gentle agitation. Then, 40 mL of Protein A BmAgo3 with an N-terminal FLAG/His-tag sequence was ampli- Sepharose 4 Fast Flow beads (GE Healthcare Biosciences) was fied by PCR with the gene-specific primers (Supplemental Table added to the lysate and the incubation was continued for 2 h at 4) using the cDNA clone as a template. The PCR products were 4°C, followed by three washes with ice-cold RIPA buffer. cloned into the pIZ/V5-His vector, which possesses the ie2 Nucleic acids that were coimmunoprecipitated were then promoter of Orgyia pseudotsugata nucleopolyhedrovirus for the isolated using a miRVana miRNA isolation kit (Ambion) accord- constitutive expression of the gene of interest and the zeocin- ing to the manufacturer’s protocol. Briefly, the beads were resistant gene for selection of stable cell lines. BmN4 cells were suspended in 500 mL of lysis/binding buffer. Total RNA was transfected with pIZ/V5-His, pIZ/FLAG-Siwi, or FLAG-BmAgo3 extracted by equal volumes of phenol:chloroform treatment. The by using Cellfectin reagent (Invitrogen). Two days after trans- fraction of small RNA was extracted from 400 mL of total RNA fection, zeocin (final concentration, 500 mg/mL) was added to using a filter cartridge with 50 mL of preheated (95°C) elution the medium. Two weeks after drug selection, the expression of solution. Small RNAs were was loaded onto a 15% denaturing

FIGURE 4. Characterization of transposon-associated Siwi- and BmAgo3-bound piRNAs (A) The heat map indicates the number of clones of Siwi- and BmAgo3-bound piRNAs that matched to transposon sequences in the Bombyx genome. Red shows antisense clones and blue shows sense clones. (B) Strand bias of Siwi- and BmAgo3-bound piRNAs. (C) Shown is the density of individual transposon-associated piRNAs. Frequency observed in the BmN4-originated piRNAs is shown individually along the y-axis. Relative nucleotide position of each transposon is indicated along the x-axis.

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Bombyx cultured cells as a model for piRNA study

polyacrylamide gel containing 10 M urea, electrophoresed, and DmHen1, modifies germline piRNAs and single-stranded siRNAs then stained with SYBRGold. Small RNA libraries were con- in RISC. Curr Biol 17: 1265–1272. structed as described above. Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, et al. 2007. A role for Piwi and piRNAs in germ line cell maintenance Data deposition and transposon silencing in zebrafish. Cell 129: 69–82. Houwing S, Berezikov E, Ketting RF. 2008. Zili is required for germ The nucleotide sequences reported in this study have been sub- cell differentiation and meiosis in zebrafish. EMBO J 27: 2702– mitted to the DDBJ/EMBL/GenBank data bank under accession 2711. numbers of AHAAB0000001-AHAAB0547473 (BmN4-derived Hutva´gner G, McLachlan J, Pasquinelli AE, Ba´lint E, Tuschl T, piRNAs), AHAAC0000001-AHAAC1704525 (BmN4-derived Siwi- Zamore PD. 2001. A cellular function for the RNA-interference bound piRNAs), and AHAAD0000001-AHAAD2196220 (BmN- enzyme Dicer in the maturation of the let-7 small temporal RNA. derived BmAgo3-bound piRNAs). Science 293: 834–838. International Silkworm Genome Consortium. 2008. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect SUPPLEMENTAL MATERIAL Biochem Mol Biol 38: 1036–1045. Katsuma S, Tanaka S, Omuro N, Takabuchi L, Daimon T, Imanishi S, Supplemental material can be found at http://www.rnajournal.org. Yamashita S, Iwanaga M, Mita K, Maeda S, et al. 2005. Novel macula-like virus identified in Bombyx mori cultured cells. J Virol 79: 5577–5584. ACKNOWLEDGMENTS Katsuma S, Daimon T, Mita K, Shimada T. 2006. Lepidopteran ortholog of Drosophila breathless is a receptor for the baculovirus We thank Shintaro Iwasaki and Mayuko Yoda for their technical fibroblast growth factor. J Virol 80: 5474–5481. assistance. This work was supported by grants from MEXT (No. Kawaoka S, Minami K, Katsuma S, Mita K, Shimada T. 2008a. 17018007 to T.S. and No. 19688004 to S.K. [last author]), MAFF- Developmentally synchronized expression of two Bombyx mori Piwi subfamily genes, SIWI and BmAGO3 in germ-line cells. NIAS (Agrigenome Research Program), and JST (Professional Biochem Biophys Res Commun 367: 755–760. Program for Agricultural Bioinformatics), Japan. Kawaoka S, Hayashi N, Katsuma S, Kishino H, Kohara Y, Mita K, Shimada T. 2008b. Bombyx small RNAs: Genomic defense system Received November 4, 2008; accepted April 14, 2009. against transposons in the silkworm, Bombyx mori. Insect Biochem Mol Biol 38: 1058–1065. Kirino Y, Mourelatos Z. 2007. Mouse Piwi-interacting RNAs are REFERENCES 29-O-methylated at their 39 termini. Nat Struct Mol Biol 14: 347– 348. Aravin AA, Sachidanandam R, Bourc’his D, Schaefer C, Pezic D, Klattenhoff C, Theurkauf W. 2008. Biogenesis and germline functions Toth KF, Bestor T, Hannon GJ. 2008. A piRNApathway primed by of piRNAs. Development 135: 3–9. individual transposons is linked to de novo DNA methylation in Li R, Li Y, Kristiansen K, Wang J. 2008. SOAP: Short oligonucleotide mice. Mol Cell 31: 785–799. alignment program. Bioinformatics 24: 713–714. Bently DR. 2006. Whole-genome re-sequencing. Curr Opin Genet Dev O’Donell KA, Boeke JD. 2007. Mighty Piwis defend the germline 16: 545–552. against genome intruders. Cell 129: 37–44. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. 2001. Role for a Ohara T, Sakaguchi Y, Suzuki T, Ueda H, Miyauchi K, Suzuki T. 2007. bidentate ribonuclease in the initiation step of RNA interference. The 39 termini of mouse Piwi-interacting RNAs are 29-O-meth- Nature 409: 363–366. ylated. Nat Struct Mol Biol 14: 349–350. Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Saito K, Sakaguchi Y, Suzuki T, Suzuki T, Siomi H, Siomi MC. 2007. Hannon GJ. 2007. Discrete small RNA-generating loci as master Pimet, the Drosophila homolog of HEN1, mediates 29-O-methyl- regulators of transposon activity in Drosophila. Cell 128: 1089– ation of Piwi- interacting RNAs at their 39 ends. Genes & Dev 21: 1103. 1603–1608. Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T. Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA. 1999. 2001. Functional anatomy of siRNAs for mediating efficient RNAi Targeted mRNA degradation by double-stranded RNA in vitro. in Drosophila melanogaster embryo lysate. EMBO J 20: 6877–6888. Genes & Dev 13: 3191–3197. Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y, Vagin VV, Signova A, Li C, Seitz H, Gvozdev V, Zamore PD. 2006. A Nagami T, Siomi H, Siomi MC. 2007. A slicer-mediated mecha- distinct small RNA pathway silences selfish genetic elements in the nism for repeat-associated siRNA 59 end formation in Drosophila. germline. Science 313: 320–324. Science 315: 1587–1590. Zamore PD. 2001. RNA intrference: Listening to the sound of silence. Hartig JV, Tomari Y, Forstemann K. 2007. piRNAs—The ancient Nat Struct Biol 8: 746–750. hunters of genome invaders. Genes & Dev 21: 1707–1713. Zamore PD, Tuschl T, Sharp PA, Bartel DP. 2000. RNAi: Double- Horwich MD, Li C, Matranga C, Vagin V, Farley G, Wang P, stranded RNA directs the ATP-dependent cleavage of mRNA at 21 Zamore PD. 2007. The Drosophila RNA methyltransferase, to 23 nucleotide intervals. Cell 101: 25–33.

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The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes

Shinpei Kawaoka, Nobumitsu Hayashi, Yutaka Suzuki, et al.

RNA published online May 21, 2009

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