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piRNAs BIOGENESIS AND ITS FUNCTIONS © 2014 Yong Huang#, Jun Yan Bai, Hong Tao Ren
Animal Science and Technology College, Henan University of Science and Technology, Luoyang City, Henan Province, 471003 P.R. China Recevied October 4, 2013; in final form November 17, 2013
piRNAs (piwi-interacting RNA) are a novel class of non-coding small single-stranded RNAs with the length of 26—33 nt. The piRNAs play important biological role through the specific interaction with the piwi proteins of the Argonaute family. piRNA function in embryonic development, maintenance of germline DNA integrity, silencing of transposon transcription, suppression of translation, formation of heterochromatin, and epigenetic regulation of sex determination. This review summarizes recent research and progress on biogenesis and function of piRNA ineukaryotic species.
Keyword: piRNA, regulation, function, cancer DOI: 10.7868/S0132342314030166
INTRODUCTION
Small regulatory RNAs have been the hot frontier of life sciences in the past few years [1]. piRNAs is a class of small non-coding RNA that was first isolated from the mouse testes and Drosophila germline independently by four groups in 2006 [2—5]. Subsequent cloning led to the discovery of numerous piRNAs [6, 7]. The length of piRNA is about 26—33 nts with a uracil deflection at 5' extremity (about 86%), and which plays an important role in spermatogenesis [8, 9]. The genes encoding piRNA are located throughout the whole genome, indicating that piRNA was present at the early stage of origin of life and has been preserved by natural species evolution [10]. piRNAs derived from single-stranded RNA precursors or two non-overlapping and divergently transcribed precursors without the involvement of Dicer endonucleases [11]. The piRNAs can be divided into three major classes based on genomic localization: non-repetitive, simple repetitive, and repeat-associated piRNAs. The non-repetative piRNAs can be further divided into three subclasses: intergenic, intronic, and exonic piRNAs. The majority of piRNA sequences (34% in the ovary and 21%, in the testes library) map to the sequences that are annotated as transposons. Similarly, more than 80% of the non-repetitive sequences can map to intergenic regions [12]. Most of the piRNAs are clustered in relatively short genome loci ranges from 1 to 100 kb [13]. All sequences, both repetitive and non-repetitive, originate from the same clusters. In turn, piRNAs in a given cluster are derived from the same orientation [13]. Establishment of piRNA database
# Corresponding author (fax: +86379 64563979; e-mail: huangyong1979111@126.com).
would facilitate scientific research of piRNA. Up to now, tens of thousands of piRNAs from drosophila, mouse, human, rat, zebrafish, and chicken have been registered in the piRNABank [14—16]. However, their function in this species is not clear. Currently, studies show that piRNAs are crucial for early development, epigenetic regulation, gametogenesis, tumorigenesis, and silencing of transposable elements (TEs), as well as some protein coding genes. In this paper, new developments related to biological functions of piRNAs are reviewed.
1. piRNA BIOGENESIS
At the core of the piRNA pathway are small RNA generative loci, piRNA clusters, which give rise to long, single-stranded transcripts, which are thought to be exported to the cytoplasm and processed into primary piRNAs [17—19]. Primary piRNAs are loaded onto piwi-related proteins and target homologous sequences for cleavage and generation of secondary piRNAs [20]. These piRNAs preferentially associate with the Argonaute proteins Piwi and Aubergine (Aub), which catalyze the generation of additional piRNAs through cleavage and processing of RNAs with antisense sequences [21, 22]. piRNA is mainly distributed in the animal testes spermatogonial cells and ovarian oocytes, and there are also a small number of piRNAs in drosophila ovarian somatic cells (follicle cells). piRNA is generated by different ways in germ cells and somatic cells (Fig. 1) [11, 23]. In somatic cells, piRNAs are produced through a PIWI-depen-dent, AUB- and AGO3-independent pathway, whereas in the germline, piRNAs are generated through an AUB- and AGO3-dependent piRNA amplification
Somatic piRNA pathway
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Fig. 1. Biogenesis of piRNAs in germline and somatic follicle cells. In the germline, piRNAs are generated through an Aub- and Ago3-dependent piRNA amplification cycle, whereas in somatic cells, biogenesis occurs through a Piwi-dependent, Aub- and Ago3-independent pathway.
cycle [24]. This amplification cycle is also called the "ping-pong" mechanism. piRNAs corresponding to the antisense strand of the retrotransposon preferentially bind Piwi/Aub protein and show a strong bias for uridine at the 5' end; sense piRNAs, by contrast, associate with Ago3 and show enrichment for adenine at position 10 [25]. Aub/Piwi cleaves transposon mRNA between positions 10 and 11 of the guide antisense piRNA, generating the 5' end of a sense Ago3-associ-ated piRNA. The mature sense piRNA is capable of guiding cleavage of the antisense transposon transcript, thus creating additional copies of the original antisense piRNA. This pathway generates a pool of piRNAs that can guide degradation of retrotransposon mRNA [25—27]. In mice, piRNAs interact with two members of the mouse Piwi proteins, Mili and Miwi2, a Piwi family protein that is required for piRNA accumulation [24, 28, 29]. Ping-pong signatures are detected mainly between Mili and Miwi2, where primary piRNAs in Mili guide slicer-mediated generation of secondary piRNAs entering Miwi2 [30]. Loading of Miwi2 licenses its nuclear entry and promotes de novo DNA methylation of transposon elements [30, 31].
2. piRNA FUNCTIONS 2.1. piRNA and Gene Silencing
The transposons are selfish DNA elements that exploit the genome and replicative machinery of host cells in order to survive and proliferate [32]. The PIWI—piRNA complexes target transposon transcripts to silence them and to protect the genomes of gametes from their invasion [33]. In ovaries, in addition to transposon transcripts, piRNAs are also derived from the 3'-untranslated region (UTR) of tj transcripts and loaded onto Piwi in OSC and OSS lines [34, 35]. It seems that the Piwi—j-derived piRNA complex functions in down-regulating particular protein-coding genes in ovarian somatic cells, such as fas-cicline III (fasIII), a cell adhesion molecule necessary for gametogenesis [34, 36]. Thus, the tj gene in ovarian somatic cells has at least dual roles regulating piwi functions: first, as a transcriptional factor that drives the piwi expression, and second, as a precursor of piRNAs that are specifically loaded onto Piwi protein to silence specific target genes, including fasIII. It should be noted that the 3'-UTRs of many other genes also act as sources for piRNAs in OSC and OSS lines [37]. Studies by Le Thomas and co-authors found that drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromo-
somes [38]. Knockdown of Piwi in the germline increases expression of transposable elements that are targeted by piRNAs, whereas protein-coding genes remain largely unaffected. Derepression of transposons upon Piwi depletion correlates with increased occupancy of Pol II on their promoters. Expression of piRNAs that target a reporter construct results in a decrease in Pol II occupancy and an increase in repressive H3K9me3 marks and heterochromatin protein 1 (HP1) on the reporter locus. Results indicate that Piwi identifies targets complementary to the associated piRNA and induces transcriptional repression by establishing a repressive chromatin state when correct targets are found [38].
2.2. piRNA and Epigenetic Regulation
The study in Drosophila implicates piRNAs in using epigenetic mechanisms to exert their gene expression effects [39]. DNA methylation is an epigenetic silencing mark functionally linked to Piwi. Mouse Piwi proteins Mili and Miwi2 are also shown to regulate DNA methylation at transposon loci [40—42]. These works imply that piRNA—Piwi pathway components may form a piRNA complex on chromatin that includes other chromatin modifying agents such as heterochro-matin protein 1a (HP1a). For example, piRNAs expressed in the central nervous system (CNS) and other somatic tissues in aplysia could mediate CpG methylation and transcriptional silencing of a key plasticity-related gene, response element binding protein 2 (CREB2) [43]. It seems that piRNAs can also direct DNA methylation on non-transposon loci, such as the Rasgrf1 locus in the mouse male germline to regulate genomic imprinting and the CREB2 promoter in apl-ysia neurons to influence long-term memory plasticity [44, 45]. Recent reports showed that in C. elegansgerm line, piRNAs can initiate a multigenerational epigenetic memory of RNA that is recognizes as "non-self" [46, 47]. By using the RNA-based recognition mechanism, foreign sequences could be detected not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression. On the other hand, endogenous germline-expressed genes are actively protected from piRNA-induced silencing. This raises an intriguing possibility that mammalian small RNAs could function in epigenetic programming and generation of an epigenetic memory [48, 49]. Histone modifications are the pre-dominant means by which epigenetic information is transmitted from parents to offspring. Recent works expanded these observations by demonstrating that Piwi-piR-NAs direct the genome-wide methylation of H3 lysine 9 (H3K9me) to transcriptionally
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