The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs

Piwi-interacting RNAs (piRNAs) are a novel class of small regulatory RNAs that are expressed specifically and abundantly in germ cells. Mammalian piRNAs are 26-31 nucleotides in length and bind to Piwi proteins, but their function and biogenesis remain elusive. We previously showed that mammalian piRNAs are 2'-O-methylated at their 3' termini. The biosynthetic mechanism and function of this modification is unknown. Here, we report that the mouse homolog (mHEN1) of HEN1, a plant microRNA (miRNA) 2'-O-methyltransferase, is expressed specifically in testis and methylates 3' termini of piRNAs in vitro. These findings provide insight into the biogenesis of piRNAs.     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

Piwis and piwi-interacting RNAs in the epigenetics of cancer

An increasing body of evidence suggests that cancer cells acquire "stem-like" epigenetic and signaling characteristics during the tumorigenic process, including global DNA hypo-methylation, gene-specific DNA hyper-methylation, and small RNA deregulation. RNAs have been known to be epigenetic regulators, both in stem cells and in differentiated cells. A novel class of small RNAs, called piwi-interacting RNAs (piRNAs), maintains genome integrity by epigenetically silencing transposons via DNA methylation, especially in germline stem cells. piRNAs interact exclusively with the Piwi family of proteins. The human Piwi ortholog, Hiwi, has been found to be aberrantly expressed in a variety of human cancers and in some, its expression correlates with poor clinical prognosis. However, there has been little investigation into the potential role that Piwi and piRNAs might play in contributing to the "stem-like" epigenetic state of a cancer. This review will highlight the current evidence supporting the importance of Piwi and piRNAs in the epigenetics of cancer and provide a potential model for the role of Piwi and piRNAs in tumorigenesis.     

Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline

The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.     

Specific expression of Olpiwi1 and Olpiwi2 in medaka (Oryzias latipes) germ cells

Piwi is necessary for germ stem cell survival in Drosophila and homologues have been identified in a diverse range of organisms. Here, we identify and characterize two homologous genes of piwi, Olpiwi1 and Olpiwi2, in the model fish medaka (Oryzias latipes). Olpiwi1 is similar to Ziwi in zebrafish or Miwi in the mouse, and Olpiwi2 is similar to Zili in zebrafish or Mili in the mouse. Moreover, Olpiwi2 mRNA is produced from two different chromosomes. RT-PCR showed expression of Olpiwi1 and Olpiwi2 predominantly in the gonads. In situ hybridization revealed germ cell-specific expression of Olpiwi1 and Olpiwi2 throughout the development of oocytes from oogonia to mature oocytes in the ovary, and from spermatogonia to spermatocytes in the testes of adults. RT-PCR and whole mount in situ hybridization showed that both Olpiwi1 and Olpiwi2 were maternally deposited in the embryo. Olpiwi1 and Olpiwi2 were detected in primordial germ cells during embryonic development. These results suggest that both Olpiwi1 and Olpiwi2 are germ cell specific, and may play important roles in germ cell development and gametogenesis in this model species.     

PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans

In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.     

Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are "specialised" in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.     

The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells

Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways.     

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.     

Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud¹ mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.