Molecular Cell

Review Posttranscriptional Regulation of Gene Expression by Piwi Proteins and piRNAs Toshiaki Watanabe1,* and Haifan Lin1,* 1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06519, USA *Correspondence: [email protected] (T.W.), [email protected] (H.L.) http://dx.doi.org/10.1016/j.molcel.2014.09.012

Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for gametogenesis, embryogenesis, and stem cell maintenance in animals. Piwi proteins act on transposon RNAs by cleaving the RNAs and by interacting with factors involved in RNA regulation. Additionally, piRNAs generated from transposons and psuedogenes can be used by Piwi proteins to regulate mRNAs at the posttranscriptional level. Here we discuss piRNA biogenesis, recent findings on posttranscriptional regulation of mRNAs by the piRNA pathway, and the potential importance of this posttranscriptional regulation for a variety of biological processes such as gametogenesis, developmental transitions, and sex determination. Introduction In eukaryotes, there are three major classes of small RNAs involved in posttranscriptional regulation: microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNA) (Kim et al., 2009; Malone and Hannon, 2009). All three types of small RNAs bind to Argonaute family proteins, which consist of the Argonaute and Piwi subfamilies. miRNAs and siRNAs bind to proteins in the Argonaute subfamily, whereas piRNAs bind to the Piwi subfamily of proteins. All three types of small RNAs find target RNAs by base pairing between complementary sequences, causing target RNA degradation and/ or translational repression (Filipowicz et al., 2008; Rana, 2007). Each type of small RNA has its own preferred class of RNA targets, reflecting their functional emphasis and mechanisms of biogenesis. For example, siRNAs generated from viral dsRNAs regulate RNA viruses (Ding and Voinnet, 2007). In contrast, piRNAs are important in transposon silencing (Bortvin, 2013; Malone and Hannon, 2009; Mani and Juliano, 2013; Peng and Lin, 2013). piRNAs are generated from various portions of long single-stranded precursor RNAs transcribed from genomic loci termed piRNA clusters, which are often >100 kb in size (Figure 1A). These piRNA clusters generally harbor many transposon sequences that have been accumulated through evolution. Once a piRNA cluster contains a transposon sequence, all other copies of this transposon elsewhere in the genome can be targeted by the piRNA pathway (Khurana et al., 2011; Sarot et al., 2004). The piRNA-mediated repression of transposons has been best characterized in the germline, where the function of the pathway appears to be the most vital. Transposons have been widely regarded as harmful parasites, yet their presence in the genome facilitates genome evolution by promoting recombination and providing the genome with functional elements (Goodier and Kazazian, 2008). To prevent excessive mutations, the host controls transposons at multiple steps of gene expression. Piwi proteins and piRNAs are involved in the degradation of transposon RNAs in the germ cells of many animals (Brennecke et al., 2007; Gunawardane et al., 2007; Reuter et al., 2011; Saito et al., 2006). In addition, they regulate transposon expression at the transcrip18 Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc.

tional level by inducing repressive epigenetic marks such as histone H3K9me3 and DNA methylation, which has been well summarized by several reviews and thus will not be the focus of this review (Castel and Martienssen, 2013; Luteijn and Ketting, 2013; Olovnikov et al., 2012; Peng and Lin, 2013; Ross et al., 2014). Beyond transcriptional regulation, a growing number of studies have suggested that piRNAs are involved in the posttranscriptional regulation of not only transposon RNAs but also other types of RNAs including mRNAs and RNA viruses (Gou et al., 2014; Kiuchi et al., 2014; Kotelnikov et al., 2009; Lim et al., 2013; Morazzani et al., 2012; Rouget et al., 2010). This is often accomplished by using transposon sequences as regulatory elements. In this review, we focus on the role of the Piwi-piRNA pathway in posttranscriptional regulation and its biological implications. To orient readers to the topic, we start with an update on piRNA biogenesis. Primary piRNA Biogenesis The Piwi subfamily proteins and piRNAs are found in diverse animals, but not in plants (Grimson et al., 2008). Our knowledge about piRNAs is based largely on studies in M. musculus, Drosophila, and C. elegans. In M. musculus and Drosophila, piRNAs are generated by two separate biogenesis pathways, primary and secondary, both of which are conserved from sponges to humans (Grimson et al., 2008), except for nematodes (Luteijn and Ketting, 2013). C. elegans piRNAs (called 21U-RNAs), 21 nt in length, are processed from
26 nt capped piRNA precursors transcribed from independent loci instead of piRNA clusters (Gu et al., 2012; Ruby et al., 2006). Furthermore, C. elegans does not possess secondary piRNA biogenesis pathway. Instead, target recognition by piRNA and PRG-1, a C. elegans Piwi protein, leads to recruitment of RNA-dependent RNA polymerase that produces siRNAs (22G-RNAs) using the target RNA as a template to reinforce silencing (Bagijn et al., 2012; Lee et al., 2012). In Drosophila, recent studies show that the HP1 homolog Rhino specifically binds to dual-strand piRNA clusters and anchors a nuclear complex that suppresses poly(A) site cleavage and splicing of the piRNA cluster transcripts (Mohn et al., 2014; Zhang et al., 2014). However, Rhino is not required for

Molecular Cell

Review A

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Primary biogenesis

Figure 1. Two Biogenesis Pathways Generate piRNAs

Secondary biogenesis (Ping-pong cycle)

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piRNA production from unistrand clusters and is not present in other animals. Presently, it is not clear how the pathway distinguishes piRNA cluster transcripts from other transcripts. Either stalled splicing (Zhang et al., 2014) or other molecular events (reviewed in Le Thomas et al., 2014) might serve as a signal for piRNA production. After transcription, the entire length of a piRNA precursor gives rise to various piRNA intermediates, though in an uneven fashion (Kawaoka et al., 2011; Saito et al., 2010; Saxe et al., 2013). The intermediates with uridine at the first nucleotide are preferentially loaded onto Piwi proteins, as determined by a loop in the middle (mid) domain of Piwi proteins (Cora et al., 2014). This enriches uracil as the first nucleotide of mature piRNAs (Kawaoka et al., 2011). Furthermore, this preferential loading partly depends on HSP90 activity (Izumi et al., 2013). However, Piwi proteins involved in secondary biogenesis (e.g., Ago3 in Drosophila, see below) do not show this preference (Brennecke et al., 2007; Gunawardane et al., 2007). Upon loading, the 30 end of the immature piRNA is trimmed by an unidentified enzyme(s), presumably until the enzyme(s) reaches the Piwi proteins (Kawaoka et al., 2011). The preferred length of piRNAs for each Piwi proteins differs even within the same species (e.g., in mice, Miwi, 2930 nt; Miwi2, 2728 nt; and Mili, 2526 nt; and in Drosophila, Piwi, 2526 nt; Aubergine, 2425 nt; and AGO3, 2324 nt), which presumably reflects the different number of nucleotides protected by each Piwi protein. Finally, the 30 ends of piRNAs are 20 -O methylated by the Hen1 methyltransferase (Horwich et al., 2007; Kirino and Mourelatos, 2007; Saito et al., 2007). In zebrafish, this modification is important for the stability of piRNAs (Kamminga et al., 2010). In Drosophila, however, the role of Hen1 in piRNA stability has been controversial (Horwich et al., 2007; Saito et al., 2007). Although several other conserved factors involved in primary biogenesis (MitoPLD/Zucchini, GASZ, GPAT2/Minotaur, and Mov10l1/Armitage) have been identified, their precise functions

Hen1 ̓ $ 2´-O-Me Secondary piRNA

(A) A model of the primary piRNA biogenesis pathway. The piRNA precursors are transcribed from piRNA clusters and are then processed into piRNA intermediates. The piRNA intermediates with uridine at the 50 ends are loaded onto Piwi proteins, with HSP90 facilitating the loading. Subsequently, the 30 portions of piRNA intermediates are trimmed by unidentified nuclease(s). After the trimming, 30 ends are 20 -O-methylated by Hen1 methyltransferase. Mitochondrial outer membrane proteins MitoPLD/Zucchini, GASZ, and GPAT2/ Minotaur are probably involved in the processing of piRNA precursors or intermediates. (B) A model of the secondary biogenesis pathway. The Piwi/piRNA complex cleaves a transposon RNA between the tenth and eleventh positions of piRNAs. The 30 region of the cleaved RNA is incorporated into Piwi proteins. The 50 region is ejected from Piwi proteins by chaperone machinery FKBP6/Shutdown and HSP90 and is then degraded. The tenth position of the incorporated RNA is enriched in adenine, because it is complementary to the first position of a piRNA that is enriched in uridine. The incorporated RNA is then processed into a mature secondary piRNA by trimming and modification, likely by the same mechanisms that generate a primary piRNA.

are still unknown. MitoPLD/Zucchini, a mitochondrial outermembrane protein, is a candidate nuclease involved in the processing of piRNA precursors or intermediates (Haase et al., 2010; Huang et al., 2011; Ipsaro et al., 2012; Murota et al., 2014; Nishimasu et al., 2012; Saito et al., 2010; Watanabe et al., 2011a). The piRNA biogenesis factors GASZ and GPAT2/Minotaur are also found in the mitochondrial outer-membrane (Czech et al., 2013; Handler et al., 2013; Ma et al., 2009; Shiromoto et al., 2013; Vagin et al., 2013), suggesting a link between the mitochondrial outer-membrane and piRNA biogenesis. Biochemical analysis is needed to determine the functions of these conserved factors in primary piRNA biogenesis. It is possible that these conserved factors regulate mRNA expression independent of Piwi proteins and piRNAs, because certain miRNA biogenesis components can regulate gene expression independent of downstream Argonaute proteins and miRNAs. For example, Drosha, an RNaseIII enzyme involved in the processing of pri-miRNA into pre-miRNA, directly downregulates mRNAs by cleaving their stem-loop structures (Han et al., 2009). Whether this happens to the piRNA pathway is worthy of investigation. Secondary piRNA Biogenesis Pathway and Transposon Suppression The secondary pathway, also known as the ping-pong cycle, selectively amplifies specific piRNAs targeting active transposons (Figure 1B; Brennecke et al., 2007; Gunawardane et al., 2007). Of the three fly Piwi proteins, Aubergine and AGO3 are involved in this process. Aubergine binds to piRNAs generated from both primary (primary piRNAs) and secondary pathways (secondary piRNAs), while AGO3 binds only to secondary piRNAs. The secondary pathway begins with cleavage of sense transposon transcripts by Aubergine bound to primary piRNAs derived from antisense transposon sequences (Figure 1B). The 30 parts of cleaved sense Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc. 19

Molecular Cell

Review B

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Figure 2. Regulation of mRNAs by piRNAs Derived from Transposons, Pseudogenes, and cis-NAT

(A) Transposon sequence-derived piRNAs regulate mRNAs. Transposon sequences in the 50 UTRs of transposon-driven mRNAs (left) and the 30 UTR of mRNAs (right) are targeted by the piRNAs. (B) Pseudogene-derived piRNAs regulate mRNAs. Pseudogenes are located in piRNA clusters in an antisense orientation to piRNA cluster transcription, so that piRNAs are produced that are antisense to the target genes. The piRNAs derived from pseudogenes then target the cognate mRNAs. (C) cis-NAT-derived piRNAs regulate sense mRNAs.

transcripts are bound to AGO3, and are processed into mature secondary piRNAs possibly by the same mechanisms in primary biogenesis (trimming and 20 -O methylation). As the slicer activity cleaves target RNAs between the tenth and eleventh positions of piRNAs, there is a 10 nt overlap between the initial Aubergine-bound primary piRNAs and the AGO3-bound secondary piRNAs generated from this process (Figure 1B). AGO3-bound secondary piRNAs in turn generate Aubergine-bound secondary piRNAs from antisense transposon transcripts by the same mechanisms. Newly generated secondary piRNAs in this process are identical to the initial primary piRNAs, and they continue to be further amplified by repeating this cycle. In silkworm BmN4 cells, the 50 parts of cleaved antisense transcripts are ejected from Ago3 proteins by chaperone machinery FKBP6/Shutdown and HSP90, and then degraded (Xiol et al., 2012). A recent study in the silkworm cell line BmN4 provided the first molecular insight into the secondary biogenesis pathway (Xiol et al., 2014). The authors propose that Vasa, a conserved DEAD box RNA helicase, promotes transfer of the target RNAs cleaved by Siwi (an Aubergine ortholog) to Ago3 for the secondary biogenesis (Xiol et al., 2014). Several proteins, including Tdrd9/Spindle-E and RNF17/Kumo, have been identified as factors involved in the secondary biogenesis (Anand and Kai, 2012; Malone et al., 2009; Shoji et al., 2009; Zhang et al., 2011). Despite this, the precise function of these factors in the secondary biogenesis remains unknown. The secondary pathway is conserved in many animals from mammals to cnidarians and probably sponges (Grimson et al., 2008; Juliano et al., 2014; Lim et al., 2014), indicating its importance for piRNA function. piRNA clusters generate primary piRNAs corresponding to many active and inactive classes of transposons. Because amplification by the secondary pathway depends on the abundance of sense and antisense transcripts, the secondary pathway selectively amplifies piRNAs derived from active transposons, resulting in efficient suppression of active transposons. The secondary pathway generates piRNAs from targeted transcripts, and therefore piRNA targets can be predicted 20 Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc.

based on the profile of secondary piRNAs. For example, 68% of Aubergine-bound piRNAs and 78% of AGO3-bound piRNAs in fly ovaries are derived from transposons and show characteristics of the secondary pathway products (Brennecke et al., 2007; Figure 1B): (1) a 10 nt overlap between a primary piRNA and a secondary piRNA (2) enrichment of A at the tenth position. These observations suggest that transposons are the main targets of Aubergine and AGO3 in fly ovaries. In fact, piRNA pathway mutations in fly ovaries increase the expression of many transposons, but do not alter mRNA expression (Klattenhoff et al., 2009; Li et al., 2009). However, a growing number of studies outside the fly ovary have revealed that piRNAs are involved in posttranscriptional regulation of mRNAs, which will be the focus of the remainder of this review. mRNA Regulation by piRNAs Derived from Transposons Posttranscriptional regulation of mRNAs by piRNAs is often mediated by the transposon sequences in mRNAs, generally in their 30 UTRs (Figure 2A; Faulkner et al., 2009). For example, more than one-quarter of mouse (27.7%) and human (28.5%) Refseqs possess at least one retrotransposon in their 30 UTRs. In zebrafish, Xenopus and mouse oocytes, Piwi proteins, and piRNAs are mainly found in the cytoplasm, and they are involved in the suppression of retrotransposons (Houwing et al., 2007; Lau et al., 2009; Lim et al., 2013; Watanabe et al., 2008). These observations suggest that retrotransposons are suppressed by piRNA pathway posttranscriptionally in vertebrate oocytes. In mouse oocytes, insertion of transposon sequences confers instability to reporter mRNAs (Watanabe et al., 2006). A recent study revealed that some transposon-driven mRNAs harboring transposon sequences in their 50 UTRs are upregulated in Mvh, Mili, and Gasz mutant oocytes (Lim et al., 2013). These observations suggest that these transposon-driven mRNAs are directly regulated by piRNAs through the transposon sequences in their 50 UTRs. Similarly, targeting of 30 UTR transposon sequences by piRNAs has been reported in the fly early embryo (see below) (Rouget et al., 2010).

Molecular Cell

Review mRNA Regulation by piRNAs Derived from Pseudogenes and cis-Natural Antisense Transcripts In the fly testis, the X-linked Stellate gene is suppressed by a pseudogene on the Y-chromosome, termed Su(Ste) (Kotelnikov et al., 2009; Livak, 1984; Figure 2B). In the absence of Su(Ste), the Stellate gene product is accumulated and forms a crystal structure in spermatocytes, which leads to infertility. The Su(Ste) locus produces piRNAs that target Stellate mRNAs for degradation (Kotelnikov et al., 2009). Notably, 70% of Aubergine-associated piRNAs in the fly testis are Su(Ste) piRNAs that target sense Stellate mRNAs (Nagao et al., 2010), suggesting that Stellate mRNAs are a main target of Aubergine in fly testes. Pseudogene-derived piRNAs have been also reported by a recent study in the adult marmoset testis (Hirano et al., 2014). In addition to trans-natural antisense transcripts (trans-NATs) from pseudogenes, cis-natural antisense transcripts (cis-NATs), transcribed from the opposite strand of the endogenous genes, have the potential to produce piRNAs that target mRNAs (Figure 2C). A recent comparative study between two Drosophila strains showed that novel piRNA clusters are formed around a number of the recently transposon-inserted loci (Shpiz et al., 2014). In a few cases, production of piRNAs from the inserted transposons spreads into coding and UTR regions, which leads to the production of piRNAs complementary to the mRNAs. These piRNAs may affect the mRNA expression posttranscriptionally and/or transcriptionally. Biological Functions of piRNA-Mediated Posttranscriptional Regulation Sex Determination by a Single piRNA A recent study in silkworms has revealed that a single piRNA from a sex chromosome plays an important role in sex determination (Kiuchi et al., 2014; Figure 3A). Silkworms use a ZW sex determination system, with males having two Z chromosomes and females having Z and W chromosomes. The sex-determining region of the W chromosome produces a precursor of the sex-determining piRNA (fem piRNA), and therefore fem piRNA is specifically expressed in females. This piRNA downregulates Masc mRNA, which encodes a CCCH-type zinc finger protein inducing masculinization. This regulation likely occurs, at least in part, at the posttranscriptional level, since Masc mRNA is cleaved at the predicted target site of fem piRNA. Inhibition of fem piRNA using antisense RNA causes femaleto-male sex reversal. These observations suggest that fem piRNA from the sex chromosome acts as a master regulator of feminization. The sex-determination role of fem piRNA indicates that a single piRNA can cause feminization. However, fem piRNA is likely abundantly expressed owing to multiple copies of its precursor on the W chromosome and the ping-pong cycle between its precursor and the target mRNA (Masc mRNA). This indicates that a high level of expression is necessary for a single piRNA to obtain efficient repression. Similarly, the Su(Ste) locus in flies contains multiple Stellate pseudogenes on the Y chromosome (Kotelnikov et al., 2009; Livak, 1984). For amplification by the ping-pong cycle, >40 nt (2 3 piRNA length  10) of complementarity is needed between precursors and target RNAs. It would be interesting to know how the complementarity between the

fem piRNA precursor and Masc mRNA has arisen during evolution, as this long stretch of complementarity could not have arisen by chance. Function of Pachytene piRNAs in mRNA Elimination Mammalian adult testes express a large amount of piRNAs, called pachytene piRNAs, which begin their expression at the pachytene spermatocyte stage and are mostly produced via primary biogenesis pathway and less dense in active transposon sequences (Figure 3B) (Beyret and Lin, 2011; Bortvin, 2013). Pachytene piRNAs are mainly bound by Miwi, and they are highly expressed from the late spermatocyte stage to the early round spermatid stage. In Miwi KO mice, spermatogenesis is arrested at the early round spermatid stage (Deng and Lin, 2002). These observations suggest that Miwi and pachytene piRNAs have an important function in late spermatocytes and early round spermatids. Although the L1 transposon is regulated by Miwi and pachytene piRNAs (Reuter et al., 2011), active transposon piRNAs including L1 piRNAs constitute only a small fraction of the pachytene piRNA population. Furthermore, increase of L1 expression by deletion of a part of a pachytene piRNA cluster has no impact on spermatogenesis (Xu et al., 2008). These observations strongly suggest that pachytene piRNAs have additional functions beyond L1 transposon suppression. A recent study reported that pachytene piRNAs and Miwi indeed have an additional role in the later stage of spermatogenesis (Gou et al., 2014). During spermiogenesis, mRNAs are globally eliminated at the elongating spermatid stage, and mature sperm retain few mRNAs. Miwi selectively assembles with the deadenylase CAF1, a catalytic component of the CCR4-Not deadenylase complex, at the elongating spermatid stage for massive mRNA elimination (Gou et al., 2014). While it is unclear how Miwi selectively associates with CAF1 only during this stage of spermatogenesis, there are data that support the functional importance of this connection. Knockdown of Miwi causes upregulation of >40% of the mRNAs in elongating spermatids, and most of them are also upregulated in the CAF1 knockdown. Furthermore, inhibition of two piRNAs using antisense oligonucleotides resulted in increases of their predicted target mRNAs in elongating spermatids. During spermiogenesis, these target mRNAs are deadenylated and sharply decreased in elongating spermatids, while neither deadenylation nor a decrease in mRNA level was observed for Gapdh, which lacks piRNA target sites. These observations suggest that Miwi and pachytene piRNAs may induce massive mRNA deadenylation and elimination in elongating spermatids. Maternal mRNA Decay by piRNAs Maternal mRNAs are largely replaced by zygotically transcribed mRNAs during the maternal-to-zygotic transition. A study in Drosophila reported that piRNAs mediate the decay of maternal nanos mRNA during the maternal-to-zygotic transition (Rouget et al., 2010). Decay of the nanos mRNA during the transition involves deadenylation by the CCR4-Not deadenylase complex. In the Aubergine mutant embryo, deadenylation and degradation of nanos mRNA is impaired. Within the nanos mRNA, two transposon fragment sequences, which are putative piRNA target sequences, are necessary for deadenylation and decay, suggesting that piRNAs in complex with Aubergine may target the transposon sequences. Aubergine forms a complex with Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc. 21

Molecular Cell

Review A W-chromosome piRNA cluster

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Figure 3. Biological Functions of Piwi Proteins and piRNAs (A) Sex determination in silkworms is mediated by a single piRNA. In females, the sex determination region of the W chromosome produces the fem piRNA that degrades Masc mRNA that encodes a CCCH-type zinc finger protein. Therefore, only males can produce the Masc protein that promotes the production of the male-specific splicing variant of Bmdsx, transcription factor. Bmdsx regulates genes responsible for the sexual phenotype of the body. In the absence of Masc mRNA, the female-specific splicing variant of Bmdsx is produced. (B) Functions of pachytene piRNAs during mouse spermatogenesis. Pachytene piRNAs are mostly bound to Miwi and expressed from the late spermatocyte to the elongating spermatid stage. Spermatogenesis in the Miwi KO mouse is arrested at the early round spermatid stage. In late spermatocytes and round spermatids, Miwi and pachytene piRNAs degrade L1 RNA in a slicer activity-dependent manner. In elongating spermatids, they promote massive mRNA elimination in a slicer-independent manner by interacting with CAF1 deadenylase. (C) Maternally transmitted I element piRNAs are required for the repression of I elements in ovaries. A dysgenic cross between reactive females devoid of I elements (R strain) and inducer males carrying I elements (I strain) produces a sterile daughter (top). This daughter lacks the expression of I element piRNAs in ovaries. A nondysgenic cross between R strain males and I strain females produces a fertile daughter, which expresses I element piRNAs in ovaries (bottom). (D) piRNA-mediated RNA degradation may play an important role in the control of sporadic RNAs transcribed from open chromatin regions in the genome.

Smg, which mediates nanos mRNA deadenylation and decay by recruiting the CCR4-Not deadenylase complex (Figure 4). Thus, piRNAs and Aubergine seem to play a role in determining which RNAs are degraded during the maternal-to-zygotic transition. Smg is translated only after fertilization, and the timing of the RNA degradation seems to be determined by 22 Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc.

the expression of Smg. Several important questions remain unanswered: (1) Because Smg and CCR4 cytoplasmic distributions are globally affected in aubergine mutant embryos, many other mRNAs might be regulated by the same mechanism. Hence, is this mechanism widely used for the degradation of other maternal mRNAs? Alternatively, is the increase

Molecular Cell

Review Figure 4. Mechanisms of Regulation by Piwi Proteins and piRNAs

Slicer-independent regulation

Slicer-dependent regulation Piwi protein piRNA

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of the nanos mRNA in aubergine mutant embryos a secondary consequence of a mislocalization of Smg and CCR4 in the mutant embryo? (2) Do other animals also use the piRNA pathway for maternal mRNA decay? Answers to these questions will advance our understanding of the mechanisms of maternal-to-zygotic transition. Function of piRNAs in Hybrid Dysgenesis Hybrid dysgenesis is a phenomenon discovered in the 1970s and defined as ‘‘a syndrome of correlated genetic traits that is spontaneously induced in hybrids between certain mutually interacting strains, usually in one direction only’’ (Kidwell and Kidwell, 1976). Hybrid dysgenesis is caused by the activation of transposons, and maternally inherited piRNAs are key factors for this phenomenon (Brennecke et al., 2008; Grentzinger et al., 2012; Khurana et al., 2011). For example, crosses between female flies lacking functional I elements (termed ‘‘reactive’’ or ‘‘R’’) and male flies carrying functional I elements (termed ‘‘inducer’’ or ‘‘I’’) yield sterile female progeny due to activation of I elements in ovaries (dysgenic crosses) (Figure 3C). In contrast, no activation of I elements is observed in the progeny from the reciprocal crosses between I females and R males (nondysgenic crosses) (Figure 3C). Importantly, offspring derived from these two crosses have the same genetic information, but they inherit different maternal factors. Oocytes of I strain harbor piRNAs derived from I elements, which are amplified by secondary pathway using I element transcripts. By contrast, the R strain lacks a functional I element copy, and as a result, few I element piRNAs are present in oocytes. Therefore, I element piRNAs are transmitted maternally only in the nondysgenic cross. This difference in the amount of I element piRNAs persists from fertilization to adulthood, which results in activation of I elements in the ovaries of a dysgenic cross (Brennecke et al., 2008). Although the repressive epigenetic mark H3K9me3 induced by piRNAs might partly contribute to this phenomenon, it is likely that maternally inherited piRNAs serve as primers to initiate the secondary biogenesis pathway and concomitant posttranscriptional regulation in the nondysgenic cross. In P-M hybrid dysgenesis, which is caused by activation of inducer P element from the paternal side, restoration of fertility in aged flies has been reported (Khurana et al., 2011). Interestingly, in the ovary of these aged flies, de novo integration of transposons into piRNA clusters is observed, indicating that

Smg

Ccr4/Caf1/Not complex

The left figure shows a model of slicer-dependent target RNA degradation. The slicer activity of Piwi proteins cleaves target RNAs. For the cleavage, near-perfect complementarity is needed between a target RNA and a piRNA. The 50 fragment of the cleaved RNA is probably degraded by 30 /50 exonucleases. The 30 fragment is likely either degraded by 30 /50 exonucleases or processed into secondary piRNAs. The right figure shows possible mechanisms of slicer-independent regulation. For this, extensive complementarity is probably not needed. All proteins in this figure have been shown to interact or colocalize with Piwi proteins. They are involved in RNA degradation (XRN1), decapping (DCP1/2), translation initiation (cytoplasmic capbinding complex), deadenylation (Caf1 and Ccr4/ Caf1/Not complex), and RNA binding (Smg).

acquisition of transposon sequence in piRNA clusters and piRNA production from the inserted sequence contribute to the restoration of fertility. Thus, piRNA pathway is a highly adaptive system to counteract transposon invasion. Expansion of piRNA clusters in the genome could increase the chance of transposon integration into piRNA clusters and therefore increase adaptation efficiency. This may explain why piRNA clusters are large and why some piRNA clusters (e.g., pachytene piRNA) retain apparently nonfunctional sequences that do not show complementarity to other regions of the genome. Function of piRNAs in Clearance of Intergenic Transcripts from Open Chromatin During gametogenesis, epigenetic information is globally reprogrammed. In mice, Piwi proteins and piRNAs are highly expressed in two phases: the gonocyte stage and the pachytene spermatocyte to round spermatid stage. Interestingly, low levels of repressive epigenetic marks are specifically observed at these stages. At the early gonocyte stage, DNA methylation is almost completely absent and becomes re-established at the late gonocyte stage (Sasaki and Matsui, 2008). In pachytene spermatocytes, H3K9me1/2/3 marks are dramatically decreased (Liu et al., 2010; Tachibana et al., 2007). In late spermatocytes and early round spermatids, several histone variants associated with open chromatin structure replace standard histones (Kimmins and Sassone-Corsi, 2005). In these cells, long noncoding RNAs (lncRNAs) are pervasively expressed from intergenic regions, probably due to open chromatin structure (Soumillon et al., 2013). These lncRNAs are possible targets of piRNAs, since retrotransposon sequences are frequently observed in lncRNAs (66% of mouse lincRNAs contain at least one transposable element) (Kelley and Rinn, 2012). Indeed, a retrotransposon sequence in lncRNA transcribed from Rasgrf1-imprinted locus is targeted by piRNAs for degradation (Watanabe et al., 2011b). In addition, in mouse round spermatids, some lncRNAs are enriched in chromatoid body, a probable site of Piwi protein and piRNA function (discussed in the next section) (Meikar et al., 2014). Given the coincidence of mouse piRNA expression and open chromatin, it is tempting to speculate that Piwi and piRNAs are expressed in these stages in order to control lncRNAs, retrotransposon RNAs, and mRNAs unnecessarily transcribed from open chromatin (Figure 3D). Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc. 23

Molecular Cell

Review Mechanisms of piRNA-Mediated Posttranscriptional Regulation The mechanistic details of posttranscriptional regulation by piRNAs are poorly understood, in contrast to miRNA and siRNA-mediated regulation. The modes of regulation by the miRNA and siRNA pathways differ among cell types, organisms, and partner Argonaute proteins. In the miRNA and siRNA pathways, Argonaute family proteins exert their repressive function by using slicer activity and/or by slicer-independent mechanisms such as inhibition of eIF4E cap binding and recruitment of other proteins involved in translational repression and RNA degradation (Filipowicz et al., 2008; Rana, 2007). High complementarity between miRNA/siRNA and their target RNAs is usually required for the slicing, and sliced target RNAs are degraded by general RNA degradation machinery such as XRN1 (50 /30 exonuclease), exosomes (30 /50 exonuclease and endonuclease) and Ski complexes (30 /50 exonuclease) (Orban and Izaurralde, 2005). Nonsliced target RNAs are translationally repressed or degraded in P bodies, cytoplasmic foci involved in translational repression, decapping, and degradation of unwanted mRNAs (Filipowicz et al., 2008). The involvement of Piwi proteins and piRNAs in the degradation of target RNAs is supported by the observation that mutating cytoplasmic Piwi proteins usually results in upregulation of transposon RNAs. (Houwing et al., 2008; Reuter et al., 2011; Vagin et al., 2006; Watanabe et al., 2008). Although there is no convincing evidence indicating that piRNAs are involved in translational repression, some observations indicate that target mRNAs might be translationally repressed by Piwi proteins and piRNAs. For example, Miwi and Mili have been reported to interact with the cytoplasmic mRNA cap binding complex in the mouse testis (Figure 4; Grivna et al., 2006; Unhavaithaya et al., 2009). In the Drosophila testis, a greater extent of protein upregulation than mRNA upregulation of the Stellate gene has been observed in the Aubergine mutant (Kotelnikov et al., 2009). Both slicer and slicer-independent mechanisms have been reported for degradation of target RNAs by Piwi proteins and piRNAs (Figure 4). For example, in mouse late spermatocytes and round spermatids, L1 RNA degradation by Miwi is dependent on its slicer activity. Furthermore, the Miwi slicer mutant and the Miwi KO mice show the same spermatogenic arrest phenotype at the early round spermatid stage (Figure 3B; Reuter et al., 2011). For efficient cleavage by the Miwi slicer activity, base pairing of nucleotides 2–22 in the piRNA with its target was shown to be required in vitro for two representative piRNAs (Reuter et al., 2011). In elongating spermatids (Figure 3B), Miwi has been reported to assemble with CAF1 deadenylase and causes deadenylation and decay of target mRNAs without slicer activity (Gou et al., 2014). Extensive base-pairing between piRNAs and target RNAs does not seems to be required for this regulation, as there are many mismatches between putative target mRNAs and piRNAs. In the germline of many organisms, Piwi proteins are usually localized to cellular organelle called the nuage, which is also known as intermitochondrial cement or the chromatoid body (Chuma et al., 2009; Pek et al., 2012). The nuage/intermitochondrial cement is a non-membrane-bound cytoplasmic electron-dense ribonucleoprotein complex usually accompanied by 24 Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc.

mitochondria and/or associated with the nuclear envelope. The nuage/intermitochondrial cement also contains several other piRNA pathway components involved in primary biogenesis and, in many cases, secondary biogenesis, and therefore is thought to be involved in the biogenesis and function of piRNAs (Chuma et al., 2009; Meikar et al., 2014; Pek et al., 2012). piRNA precursors are enriched in chromatoid body in mouse spermatids (Meikar et al., 2014). However, the initial steps of primary biogenesis probably occur on the mitochondrial surface, where MitoPLD/Zucchini, Gasz, and GPAT2/Minotaur are localized. The nuage and chromatoid body might be involved in the final steps of primary biogenesis and the secondary biogenesis, both of which involve loading of piRNAs onto Piwi proteins, trimming of 30 ends of piRNA intermediates, and methylation of 30 ends of mature piRNAs. A recent study in Planarian shows that Histone H4 mRNA is localized to chromatoid body in a manner dependent on Piwi proteins (Rouhana et al., 2014). Furthermore, this mRNA is mildly upregulated upon knockdown of Piwi proteins. Because this mRNA produces only a small number of piRNAs, it is not likely to be a piRNA precursor. Examining whether this mRNA is targeted by piRNA should aid the understanding of the function of the chromatoid body. In addition to nuage and chromatoid localization, some Piwi proteins are also localized to P bodies or colocalized with P body components (Aravin et al., 2009; Lim et al., 2009; Liu et al., 2011; Tanaka et al., 2011). For example, a study in the Drosophila ovary showed that a small fraction of Aubergine is found in P bodies, and transposon transcripts are localized to P bodies in a manner dependent on Aubergine (Lim et al., 2009). Therefore, it is possible that P body components contribute to the degradation of transcripts targeted by Aubergine (Figure 4). In mouse fetal testes, Miwi2 is localized to P bodies (Aravin et al., 2009; Shoji et al., 2009), and this localization is abrogated in mice defective in piRNA biogenesis (Aravin et al., 2009; Saxe et al., 2013; Watanabe et al., 2011a), suggesting that piRNAs are required for localization of Miwi2 into the P body. Given that the Miwi2 slicer activity has been shown to be dispensable for its function (De Fazio et al., 2011), it is possible Miwi2 facilitates target RNA degradation independent of its slicer activity using P body components (Figure 4). Perspectives The piRNA-mediated posttranscriptional regulation has just begun to be explored. Important questions include the following: Do Piwi proteins have a more general role in promoting the degradation of target RNAs beyond transposons outside the mouse adult testis? Is this role independent of their slicer activity? If so, what is the slicer-independent mechanism responsible for target RNA degradation? Also, is the expression of target RNAs repressed at the translational level? If so, what is the molecular mechanism of repression? What are the roles of different cellular structures, such as the nuage, the chormatoid body, and P bodies, in piRNA-mediated regulation? Answers to these questions will significantly advance our understanding of posttranscriptional regulation by the Piwi-piRNA pathway. In addition, given that hundreds of thousands of different piRNAs are expressed in an individual cell, there may be more stringent rules for interactions between piRNAs and target RNAs as

Molecular Cell

Review compared to the miRNA pathway in order to avoid frequent off-target effects. Exploring the general rules of interactions between piRNAs and their target RNAs for slicer-mediated cleavage and for slicer-independent regulation will be important for precise target prediction. In many organisms, mutations in Piwi proteins reveal that the Piwi-piRNA pathway regulates important biological processes such as fertility, gametogenesis, cell viability, and stem cell maintenance, yet mechanisms underlying this regulation remain mostly unknown. It is likely that Piwi proteins and piRNAs suppress the expression of transposons to prevent defects caused by overexpression of transposons. However, how and how much increased transposon expression contributes to cell death or other defects remain to be answered. Although piRNAs are involved in posttranscriptional transposon suppression in mouse oocytes, oocytes mutant for the Piwi-piRNA pathway do not show any visible defects (Lim et al., 2013; Watanabe et al., 2008). Presumably, the consequence of failed suppression in the mutants may only become apparent in future generations. Finally, the transposon-related effect is likely only part of the piRNA function, because it is now becoming clear that many mRNAs are also regulated by piRNAs. Moreover, given the coincidence of piRNA expression and widespread lncRNA expression in mice, piRNA may function in lncRNA regulation. Therefore, misregulation of mRNAs and lncRNAs may contribute to the observed phenotypes. ACKNOWLEDGMENTS We thank Celina Juliano and Sean Christensen for comments on the manuscript. Current research in the Lin lab on Piwi proteins and piRNA is supported by the NIH (DP1CA174418 and R01HD42012), the G. Harold & Leila Mathers Foundation, and an Ellison Medical Foundation Senior Scholar Award.

REFERENCES Anand, A., and Kai, T. (2012). The tudor domain protein kumo is required to assemble the nuage and to generate germline piRNAs in Drosophila. EMBO J. 31, 870–882. Aravin, A.A., van der Heijden, G.W., Castan˜eda, J., Vagin, V.V., Hannon, G.J., and Bortvin, A. (2009). Cytoplasmic compartmentalization of the fetal piRNA pathway in mice. PLoS Genet. 5, e1000764. Bagijn, M.P., Goldstein, L.D., Sapetschnig, A., Weick, E.M., Bouasker, S., Lehrbach, N.J., Simard, M.J., and Miska, E.A. (2012). Function, targets, and evolution of Caenorhabditis elegans piRNAs. Science 337, 574–578. Beyret, E., and Lin, H. (2011). Pinpointing the expression of piRNAs and function of the PIWI protein subfamily during spermatogenesis in the mouse. Dev. Biol. 355, 215–226. Bortvin, A. (2013). PIWI-interacting RNAs (piRNAs) - a mouse testis perspective. Biochemistry Mosc. 78, 592–602. Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R., and Hannon, G.J. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103. Brennecke, J., Malone, C.D., Aravin, A.A., Sachidanandam, R., Stark, A., and Hannon, G.J. (2008). An epigenetic role for maternally inherited piRNAs in transposon silencing. Science 322, 1387–1392. Castel, S.E., and Martienssen, R.A. (2013). RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat. Rev. Genet. 14, 100–112.

Chuma, S., Hosokawa, M., Tanaka, T., and Nakatsuji, N. (2009). Ultrastructural characterization of spermatogenesis and its evolutionary conservation in the germline: germinal granules in mammals. Mol. Cell. Endocrinol. 306, 17–23. Cora, E., Pandey, R.R., Xiol, J., Taylor, J., Sachidanandam, R., McCarthy, A.A., and Pillai, R.S. (2014). The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs. RNA 20, 773–781. Czech, B., Preall, J.B., McGinn, J., and Hannon, G.J. (2013). A transcriptomewide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway. Mol. Cell 50, 749–761. De Fazio, S., Bartonicek, N., Di Giacomo, M., Abreu-Goodger, C., Sankar, A., Funaya, C., Antony, C., Moreira, P.N., Enright, A.J., and O’Carroll, D. (2011). The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements. Nature 480, 259–263. Deng, W., and Lin, H. (2002). miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev. Cell 2, 819–830. Ding, S.W., and Voinnet, O. (2007). Antiviral immunity directed by small RNAs. Cell 130, 413–426. Faulkner, G.J., Kimura, Y., Daub, C.O., Wani, S., Plessy, C., Irvine, K.M., Schroder, K., Cloonan, N., Steptoe, A.L., Lassmann, T., et al. (2009). The regulated retrotransposon transcriptome of mammalian cells. Nat. Genet. 41, 563–571. Filipowicz, W., Bhattacharyya, S.N., and Sonenberg, N. (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102–114. Goodier, J.L., and Kazazian, H.H., Jr. (2008). Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell 135, 23–35. Gou, L.T., Dai, P., Yang, J.H., Xue, Y., Hu, Y.P., Zhou, Y., Kang, J.Y., Wang, X., Li, H., Hua, M.M., et al. (2014). Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis. Cell Res. 24, 680–700. Grentzinger, T., Armenise, C., Brun, C., Mugat, B., Serrano, V., Pelisson, A., and Chambeyron, S. (2012). piRNA-mediated transgenerational inheritance of an acquired trait. Genome Res. 22, 1877–1888. Grimson, A., Srivastava, M., Fahey, B., Woodcroft, B.J., Chiang, H.R., King, N., Degnan, B.M., Rokhsar, D.S., and Bartel, D.P. (2008). Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature 455, 1193– 1197. Grivna, S.T., Pyhtila, B., and Lin, H. (2006). MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis. Proc. Natl. Acad. Sci. USA 103, 13415–13420. Gu, W., Lee, H.C., Chaves, D., Youngman, E.M., Pazour, G.J., Conte, D., Jr., and Mello, C.C. (2012). CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors. Cell 151, 1488– 1500. Gunawardane, L.S., Saito, K., Nishida, K.M., Miyoshi, K., Kawamura, Y., Nagami, T., Siomi, H., and Siomi, M.C. (2007). A slicer-mediated mechanism for repeat-associated siRNA 50 end formation in Drosophila. Science 315, 1587–1590. Haase, A.D., Fenoglio, S., Muerdter, F., Guzzardo, P.M., Czech, B., Pappin, D.J., Chen, C.F., Gordon, A., and Hannon, G.J. (2010). Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila. Genes Dev. 24, 2499–2504. Han, J., Pedersen, J.S., Kwon, S.C., Belair, C.D., Kim, Y.K., Yeom, K.H., Yang, W.Y., Haussler, D., Blelloch, R., and Kim, V.N. (2009). Posttranscriptional crossregulation between Drosha and DGCR8. Cell 136, 75–84. Handler, D., Meixner, K., Pizka, M., Lauss, K., Schmied, C., Gruber, F.S., and Brennecke, J. (2013). The genetic makeup of the Drosophila piRNA pathway. Mol. Cell 50, 762–777. Hirano, T., Iwasaki, Y.W., Lin, Z.Y., Imamura, M., Seki, N.M., Sasaki, E., Saito, K., Okano, H., Siomi, M.C., and Siomi, H. (2014). Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset, a model primate. RNA 20, 1223–1237.

Molecular Cell 56, October 2, 2014 ª2014 Elsevier Inc. 25

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Review Horwich, M.D., Li, C., Matranga, C., Vagin, V., Farley, G., Wang, P., and Zamore, P.D. (2007). The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Current Biol. 17, 1265– 1272. Houwing, S., Kamminga, L.M., Berezikov, E., Cronembold, D., Girard, A., van den Elst, H., Filippov, D.V., Blaser, H., Raz, E., Moens, C.B., et al. (2007). A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129, 69–82. Houwing, S., Berezikov, E., and Ketting, R.F. (2008). Zili is required for germ cell differentiation and meiosis in zebrafish. EMBO J. 27, 2702–2711. Huang, H.Y., Gao, Q., Peng, X.X., Choi, S.Y., Sarma, K., Ren, H.M., Morris, A.J., and Frohman, M.A. (2011). piRNA-associated germline nuage formation and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling. Dev. Cell 20, 376–387. Ipsaro, J.J., Haase, A.D., Knott, S.R., Joshua-Tor, L., and Hannon, G.J. (2012). The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283. Izumi, N., Kawaoka, S., Yasuhara, S., Suzuki, Y., Sugano, S., Katsuma, S., and Tomari, Y. (2013). Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins. RNA 19, 896–901. Juliano, C.E., Reich, A., Liu, N., Go¨tzfried, J., Zhong, M., Uman, S., Reenan, R.A., Wessel, G.M., Steele, R.E., and Lin, H. (2014). PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells. Proc. Natl. Acad. Sci. USA 111, 337–342. Kamminga, L.M., Luteijn, M.J., den Broeder, M.J., Redl, S., Kaaij, L.J.T., Roovers, E.F., Ladurner, P., Berezikov, E., and Ketting, R.F. (2010). Hen1 is required for oocyte development and piRNA stability in zebrafish. EMBO J. 29, 3688–3700. Kawaoka, S., Izumi, N., Katsuma, S., and Tomari, Y. (2011). 30 end formation of PIWI-interacting RNAs in vitro. Mol. Cell 43, 1015–1022. Kelley, D., and Rinn, J. (2012). Transposable elements reveal a stem cell-specific class of long noncoding RNAs. Genome Biol. 13, R107. Khurana, J.S., Wang, J., Xu, J., Koppetsch, B.S., Thomson, T.C., Nowosielska, A., Li, C., Zamore, P.D., Weng, Z., and Theurkauf, W.E. (2011). Adaptation to P element transposon invasion in Drosophila melanogaster. Cell 147, 1551– 1563. Kidwell, M.G., and Kidwell, J.F. (1976). Selection for male recombination in Drosophila melanogaster. Genetics 84, 333–351. Kim, V.N., Han, J., and Siomi, M.C. (2009). Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–139. Kimmins, S., and Sassone-Corsi, P. (2005). Chromatin remodelling and epigenetic features of germ cells. Nature 434, 583–589.

Lee, H.C., Gu, W., Shirayama, M., Youngman, E., Conte, D., Jr., and Mello, C.C. (2012). C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts. Cell 150, 78–87. Li, C., Vagin, V.V., Lee, S., Xu, J., Ma, S., Xi, H., Seitz, H., Horwich, M.D., Syrzycka, M., Honda, B.M., et al. (2009). Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137, 509–521. Lim, A.K., Tao, L., and Kai, T. (2009). piRNAs mediate posttranscriptional retroelement silencing and localization to pi-bodies in the Drosophila germline. J. Cell Biol. 186, 333–342. Lim, A.K., Lorthongpanich, C., Chew, T.G., Tan, C.W., Shue, Y.T., Balu, S., Gounko, N., Kuramochi-Miyagawa, S., Matzuk, M.M., Chuma, S., et al. (2013). The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles. Development 140, 3819–3825. Lim, R.S., Anand, A., Nishimiya-Fujisawa, C., Kobayashi, S., and Kai, T. (2014). Analysis of Hydra PIWI proteins and piRNAs uncover early evolutionary origins of the piRNA pathway. Dev. Biol. 386, 237–251. Liu, Z., Zhou, S., Liao, L., Chen, X., Meistrich, M., and Xu, J. (2010). Jmjd1a demethylase-regulated histone modification is essential for cAMP-response element modulator-regulated gene expression and spermatogenesis. J. Biol. Chem. 285, 2758–2770. Liu, L., Qi, H., Wang, J., and Lin, H. (2011). PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition. Development 138, 1863–1873. Livak, K.J. (1984). Organization and mapping of a sequence on the Drosophila melanogaster X and Y chromosomes that is transcribed during spermatogenesis. Genetics 107, 611–634. Luteijn, M.J., and Ketting, R.F. (2013). PIWI-interacting RNAs: from generation to transgenerational epigenetics. Nat. Rev. Genet. 14, 523–534. Ma, L., Buchold, G.M., Greenbaum, M.P., Roy, A., Burns, K.H., Zhu, H.F., Han, D.Y., Harris, R.A., Coarfa, C., Gunaratne, P.H., et al. (2009). GASZ is essential for male meiosis and suppression of retrotransposon expression in the male germline. PLoS Genet. 5, e1000635. Malone, C.D., and Hannon, G.J. (2009). Small RNAs as guardians of the genome. Cell 136, 656–668. Malone, C.D., Brennecke, J., Dus, M., Stark, A., McCombie, W.R., Sachidanandam, R., and Hannon, G.J. (2009). Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell 137, 522–535. Mani, S.R., and Juliano, C.E. (2013). Untangling the web: the diverse functions of the PIWI/piRNA pathway. Mol. Reprod. Dev. 80, 632–664. Meikar, O., Vagin, V.V., Chalmel, F., So˜star, K., Lardenois, A., Hammell, M., Jin, Y., Da Ros, M., Wasik, K.A., Toppari, J., et al. (2014). An atlas of chromatoid body components. RNA 20, 483–495.

Kirino, Y., and Mourelatos, Z. (2007). The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs. RNA 13, 1397–1401.

Mohn, F., Sienski, G., Handler, D., and Brennecke, J. (2014). The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157, 1364–1379.

Kiuchi, T., Koga, H., Kawamoto, M., Shoji, K., Sakai, H., Arai, Y., Ishihara, G., Kawaoka, S., Sugano, S., Shimada, T., et al. (2014). A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature 509, 633–636.

Morazzani, E.M., Wiley, M.R., Murreddu, M.G., Adelman, Z.N., and Myles, K.M. (2012). Production of virus-derived ping-pong-dependent piRNA-like small RNAs in the mosquito soma. PLoS Pathog. 8, e1002470.

Klattenhoff, C., Xi, H., Li, C., Lee, S., Xu, J., Khurana, J.S., Zhang, F., Schultz, N., Koppetsch, B.S., Nowosielska, A., et al. (2009). The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dualstrand clusters. Cell 138, 1137–1149.

Murota, Y., Ishizu, H., Nakagawa, S., Iwasaki, Y.W., Shibata, S., Kamatani, M.K., Saito, K., Okano, H., Siomi, H., and Siomi, M.C. (2014). Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly. Cell Rep. 8, 103–113.

Kotelnikov, R.N., Klenov, M.S., Rozovsky, Y.M., Olenina, L.V., Kibanov, M.V., and Gvozdev, V.A. (2009). Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster. Nucleic Acids Res. 37, 3254–3263.

Nagao, A., Mituyama, T., Huang, H., Chen, D., Siomi, M.C., and Siomi, H. (2010). Biogenesis pathways of piRNAs loaded onto AGO3 in the Drosophila testis. RNA 16, 2503–2515.

Lau, N.C., Ohsumi, T., Borowsky, M., Kingston, R.E., and Blower, M.D. (2009). Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi. EMBO J. 28, 2945–2958.

Nishimasu, H., Ishizu, H., Saito, K., Fukuhara, S., Kamatani, M.K., Bonnefond, L., Matsumoto, N., Nishizawa, T., Nakanaga, K., Aoki, J., et al. (2012). Structure and function of Zucchini endoribonuclease in piRNA biogenesis. Nature 491, 284–287.

Le Thomas, A., To´th, K.F., and Aravin, A.A. (2014). To be or not to be a piRNA: genomic origin and processing of piRNAs. Genome Biol. 15, 204.

Olovnikov, I., Aravin, A.A., and Fejes Toth, K. (2012). Small RNA in the nucleus: the RNA-chromatin ping-pong. Curr. Opin. Genet. Dev. 22, 164–171.

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Molecular Cell

Review Orban, T.I., and Izaurralde, E. (2005). Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome. RNA 11, 459–469. Pek, J.W., Patil, V.S., and Kai, T. (2012). piRNA pathway and the potential processing site, the nuage, in the Drosophila germline. Dev. Growth Differ. 54, 66–77. Peng, J.C., and Lin, H. (2013). Beyond transposons: the epigenetic and somatic functions of the Piwi-piRNA mechanism. Curr. Opin. Cell Biol. 25, 190–194. Rana, T.M. (2007). Illuminating the silence: understanding the structure and function of small RNAs. Nat. Rev. Mol. Cell Biol. 8, 23–36. Reuter, M., Berninger, P., Chuma, S., Shah, H., Hosokawa, M., Funaya, C., Antony, C., Sachidanandam, R., and Pillai, R.S. (2011). Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing. Nature 480, 264–267. Ross, R.J., Weiner, M.M., and Lin, H. (2014). PIWI proteins and PIWI-interacting RNAs in the soma. Nature 505, 353–359. Rouget, C., Papin, C., Boureux, A., Meunier, A.C., Franco, B., Robine, N., Lai, E.C., Pelisson, A., and Simonelig, M. (2010). Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo. Nature 467, 1128–1132. Rouhana, L., Weiss, J.A., King, R.S., and Newmark, P.A. (2014). PIWI homologs mediate Histone H4 mRNA localization to planarian chromatoid bodies. Development 141, 2592–2601. Ruby, J.G., Jan, C., Player, C., Axtell, M.J., Lee, W., Nusbaum, C., Ge, H., and Bartel, D.P. (2006). Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207.

Soumillon, M., Necsulea, A., Weier, M., Brawand, D., Zhang, X., Gu, H., Barthes, P., Kokkinaki, M., Nef, S., Gnirke, A., et al. (2013). Cellular source and mechanisms of high transcriptome complexity in the mammalian testis. Cell Rep. 3, 2179–2190. Tachibana, M., Nozaki, M., Takeda, N., and Shinkai, Y. (2007). Functional dynamics of H3K9 methylation during meiotic prophase progression. EMBO J. 26, 3346–3359. Tanaka, T., Hosokawa, M., Vagin, V.V., Reuter, M., Hayashi, E., Mochizuki, A.L., Kitamura, K., Yamanaka, H., Kondoh, G., Okawa, K., et al. (2011). Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis. Proc. Natl. Acad. Sci. USA 108, 10579–10584. Unhavaithaya, Y., Hao, Y., Beyret, E., Yin, H., Kuramochi-Miyagawa, S., Nakano, T., and Lin, H. (2009). MILI, a PIWI-interacting RNA-binding protein, is required for germ line stem cell self-renewal and appears to positively regulate translation. J. Biol. Chem. 284, 6507–6519. Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V., and Zamore, P.D. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320–324. Vagin, V.V., Yu, Y., Jankowska, A., Luo, Y.C., Wasik, K.A., Malone, C.D., Harrison, E., Rosebrock, A., Wakimoto, B.T., Fagegaltier, D., et al. (2013). Minotaur is critical for primary piRNA biogenesis. RNA 19, 1064–1077. Watanabe, T., Takeda, A., Tsukiyama, T., Mise, K., Okuno, T., Sasaki, H., Minami, N., and Imai, H. (2006). Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev. 20, 1732– 1743.

Saito, K., Nishida, K.M., Mori, T., Kawamura, Y., Miyoshi, K., Nagami, T., Siomi, H., and Siomi, M.C. (2006). Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20, 2214–2222.

Watanabe, T., Totoki, Y., Toyoda, A., Kaneda, M., Kuramochi-Miyagawa, S., Obata, Y., Chiba, H., Kohara, Y., Kono, T., Nakano, T., et al. (2008). Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543.

Saito, K., Sakaguchi, Y., Suzuki, T., Suzuki, T., Siomi, H., and Siomi, M.C. (2007). Pimet, the Drosophila homolog of HEN1, mediates 20 -O-methylation of Piwi- interacting RNAs at their 30 ends. Genes Dev. 21, 1603–1608.

Watanabe, T., Chuma, S., Yamamoto, Y., Kuramochi-Miyagawa, S., Totoki, Y., Toyoda, A., Hoki, Y., Fujiyama, A., Shibata, T., Sado, T., et al. (2011a). MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375.

Saito, K., Ishizu, H., Komai, M., Kotani, H., Kawamura, Y., Nishida, K.M., Siomi, H., and Siomi, M.C. (2010). Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila. Genes Dev. 24, 2493–2498. Sarot, E., Payen-Groscheˆne, G., Bucheton, A., and Pe´lisson, A. (2004). Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166, 1313–1321. Sasaki, H., and Matsui, Y. (2008). Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat. Rev. Genet. 9, 129–140.

Watanabe, T., Tomizawa, S., Mitsuya, K., Totoki, Y., Yamamoto, Y., Kuramochi-Miyagawa, S., Iida, N., Hoki, Y., Murphy, P.J., Toyoda, A., et al. (2011b). Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 332, 848–852. Xiol, J., Cora, E., Koglgruber, R., Chuma, S., Subramanian, S., Hosokawa, M., Reuter, M., Yang, Z., Berninger, P., Palencia, A., et al. (2012). A role for Fkbp6 and the chaperone machinery in piRNA amplification and transposon silencing. Mol. Cell 47, 970–979.

Saxe, J.P., Chen, M.J., Zhao, H.Y., and Lin, H.F. (2013). Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline. EMBO J. 32, 1869–1885.

Xiol, J., Spinelli, P., Laussmann, M.A., Homolka, D., Yang, Z., Cora, E., Coute´, Y., Conn, S., Kadlec, J., Sachidanandam, R., et al. (2014). RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts. Cell 157, 1698–1711.

Shiromoto, Y., Kuramochi-Miyagawa, S., Daiba, A., Chuma, S., Katanaya, A., Katsumata, A., Nishimura, K., Ohtaka, M., Nakanishi, M., Nakamura, T., et al. (2013). GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells. RNA 19, 803–810.

Xu, M., You, Y., Hunsicker, P., Hori, T., Small, C., Griswold, M.D., and Hecht, N.B. (2008). Mice deficient for a small cluster of Piwi-interacting RNAs implicate Piwi-interacting RNAs in transposon control. Biol. Reprod. 79, 51–57.

Shoji, M., Tanaka, T., Hosokawa, M., Reuter, M., Stark, A., Kato, Y., Kondoh, G., Okawa, K., Chujo, T., Suzuki, T., et al. (2009). The TDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing in the mouse male germline. Dev. Cell 17, 775–787.

Zhang, Z., Xu, J., Koppetsch, B.S., Wang, J., Tipping, C., Ma, S., Weng, Z., Theurkauf, W.E., and Zamore, P.D. (2011). Heterotypic piRNA Ping-Pong requires qin, a protein with both E3 ligase and Tudor domains. Mol. Cell 44, 572–584.

Shpiz, S., Ryazansky, S., Olovnikov, I., Abramov, Y., and Kalmykova, A. (2014). Euchromatic transposon insertions trigger production of novel Pi- and endosiRNAs at the target sites in the drosophila germline. PLoS Genet. 10, e1004138.

Zhang, Z., Wang, J., Schultz, N., Zhang, F., Parhad, S.S., Tu, S., Vreven, T., Zamore, P.D., Weng, Z., and Theurkauf, W.E. (2014). The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing. Cell 157, 1353–1363.

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