Generation Of piRNAs Research Articles

Maternally inherited siRNAs initiate piRNA cluster formation

PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. Here, we report spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. We discovered that maternally supplied small interfering RNAs (siRNAs) trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs.

A chromodomain protein mediates heterochromatin-directed piRNA expression

PIWI-interacting RNAs (piRNAs) play significant roles in suppressing transposons, maintaining genome integrity, and defending against viral infections. How piRNA source loci are efficiently transcribed is poorly understood. Here, we show that in Caenorhabditis elegans, transcription of piRNA clusters depends on the chromatin microenvironment and a chromodomain-containing protein, UAD-2. piRNA clusters form distinct focus in germline nuclei. We conducted a forward genetic screening and identified UAD-2 that is required for piRNA focus formation. In the absence of histone 3 lysine 27 methylation or proper chromatin-remodeling status, UAD-2 is depleted from the piRNA focus. UAD-2 recruits the upstream sequence transcription complex (USTC), which binds the Ruby motif to piRNA promoters and promotes piRNA generation. Vice versa, the USTC complex is required for UAD-2 to associate with the piRNA focus. Thus, transcription of heterochromatic small RNA source loci relies on coordinated recruitment of both the readers of histone marks and the core transcriptional machinery to DNA.

Pachytene piRNAs as beneficial regulators or a defense system gone rogue.

Pachytene Piwi-interacting RNAs (piRNAs) are abundant small non-coding RNAs expressed in mammalian germ lines. A new study indicates that, among the diverse pool of piRNA sequences, a small number act as highly selective guides that induce cleavage of coding and non-coding transcripts, thus promoting piRNA generation and regulating gene expression.

The evolutionary arms race between transposable elements and piRNAs in Drosophila melanogaster

BackgroundThe piwi-interacting RNAs (piRNAs) are small non-coding RNAs that specifically repress transposable elements (TEs) in the germline of Drosophila. Despite our expanding understanding of TE:piRNA interaction, whether there is an evolutionary arms race between TEs and piRNAs was unclear.ResultsHere, we studied the population genomics of TEs and piRNAs in the worldwide strains of D. melanogaster. By conducting a correlation analysis between TE contents and the abundance of piRNAs from ovaries of representative strains of D. melanogaster, we find positive correlations between TEs and piRNAs in six TE families. Our simulations further highlight that TE activities and the strength of purifying selection against TEs are important factors shaping the interactions between TEs and piRNAs. Our studies also suggest that the de novo generation of piRNAs is an important mechanism to repress the newly invaded TEs.ConclusionsOur results revealed the existence of an evolutionary arms race between the copy numbers of TEs and the abundance of antisense piRNAs at the population level. Although the interactions between TEs and piRNAs are complex and many factors should be considered to impact their interaction dynamics, our results suggest the emergence, repression specificity and strength of piRNAs on TEs should be considered in studying the landscapes of TE insertions in Drosophila. These results deepen our understanding of the interactions between piRNAs and TEs, and also provide novel insights into the nature of genomic conflicts of other forms.

PiRNAs: biogenesis and their potential roles in cancer.

With the length of about 26-31nt, PIWI-interacting RNA (piRNA) is a small non-coding RNA (ncRNA) that interacts with PIWI proteins to form the piRNA silencing complex (piRISC). PIWI is a subfamily of Argonaute, and piRNA must bind to PIWI to exert its regulatory role. Current studies indicated that piRNA and PIWI are significantly abnormally expressed in gastric, breast, kidney, colon, and lung cancers, and are involved in the initiation, progression, and metastasis of cancers, which may be the potential diagnostic tools, prognostic markers, and therapeutic targets for cancers. By reviewing piRNA recent studies, this research summarized the mechanism of piRNA generation and the functions of piRNA/PIWI in gastric, breast, kidney, colon, and lung cancers, providing a reference value for further piRNA research.

Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production.

In Drosophila ovarian somatic cells, PIWI-interacting small RNAs (piRNAs) against transposable elements are mainly produced from the ∼180-kb flamenco (flam) locus. flam transcripts are gathered into foci, located close to the nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA fluorescence in situ hybridization, we found that in the follicle cells of ovaries the 5′-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, whereas their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the first and, partially, second exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNAs from the protein-coding gene transcripts accumulate at higher levels in flamKG ovaries indicating that piRNA biogenesis may occur without Yb bodies. We propose that normally in follicle cells, flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which competitively decreases piRNA production from the protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway.

PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi. Kc167 piRNAs are produced by a canonical, primary piRNA biogenesis pathway, from phased processing of precursor transcripts by the Zuc endonuclease, Armi helicase, and dGasz mitochondrial scaffold protein. Kc167 piRNAs derive from cytoplasmic transcripts, notably tRNAs and mRNAs, and their abundance correlates with that of parent transcripts. The expression of Aub is robust in Kc167, that of Piwi is modest, while Ago3 is undetectable, explaining the lack of transposon-related piRNA amplification by the Aub-Ago3, ping-pong mechanism. We propose that the default state of the primary piRNA biogenesis machinery is random transcript sampling to allow generation of piRNAs from any transcript, including newly acquired retrotransposons. This state is unmasked in Kc167, likely because they do not express piRNA cluster transcripts in sufficient amounts and do not amplify transposon piRNAs. We use Kc167 to characterize an inactive isoform of Aub protein. Since most Kc167 piRNAs are genic, they can be mapped uniquely to the genome, facilitating computational analyses. Furthermore, because Kc167 is a widely used and well-characterized cell line that is easily amenable to experimental manipulations, we expect that it will serve as an excellent system to study piRNA biogenesis and piRNA-related factors.

RNA helicase Spn-E is required to maintain Aub and AGO3 protein levels for piRNA silencing in the germline of Drosophila

Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we show that genetic inhibition of Bel function leads to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited.

Abstract A29: PIWI-like proteins and piRNAs in breast cancer cell proliferation and migration

Abstract Breast cancer is the major cause of cancer-related death in women globally. With the revelation of key molecular pathways modulating all aspects of the breast cancer, the pharmaceutical development and clinical treatment have achieved significant progress. However, patients are still threatened by serious issues, including drug resistance, relapse and metastasis. In addition, a reliable and comprehensive system of early detection of the tumor is still lacking. As such, we set out to identify potential signaling pathway that could serve as novel drug target and diagnostic indicator. Recently, the PIWI (P-element-induced wimpy testis)-like proteins are gaining more interest as prospective biomarkers and targets for therapeutic development because of their re-expression in cancer cells. Initially, the PIWI-like proteins were regarded as germline-specific regulators. They have been revealed to exert important epigenetic functions in germ cells, including the suppression of retrotransposons, the formation of heterochromatin, and the maintenance of DNA methylation status. Surprisingly, in various human tumors, the abnormal expression of PIWI-like proteins was readily observed. The in vitro data showed that the repression of these proteins significantly slowed down the tumor growth, raising an intriguing question whether the PIWI-like proteins and the associated small RNAs, piRNAs, contribute to the breast cancer initiation and progression. In this study, we investigated the expression PIWI-like proteins and piRNAs in three different breast cell lines. It was found that in the normal mammalian epithelial cell line, MCF12A, there was a general lack of PIWI-like expression, which was unaffected by the addition of estrogen. In addition, there was no indication of piRNA generation and accumulation in MCF12A. However, in two breast cancer cell lines, MCF7 and MDA-MB-231, we detected robust expression of PIWI proteins and piRNAs. The knockdown of individual PIWI-like homologs significantly impacted two important indicators of the cancer, rapid proliferation and migration capacity. Furthermore, we found that the depletion of PIWI-like homologs repressed the expression of key regulators of pro-growth genes, such as CCND1, while enhancing those anti-proliferation factors and metastasis inhibitors, such as Bcl-2, p27, p53 and Oct-4. The data indicate that PIWI-like proteins could contribute to cancer formation and progression. We will further explore whether they exert these regulatory functions at the epigenetic level, just like in germ cells. Also, a comprehensive profiling of piRNAs in breast cancer will provide further clue about the clinical potential of using the PIWI/piRNA pathway as a screening indicator. Note: This abstract was not presented at the conference. Citation Format: Jing Yi Lee, Wai Cheong Yip, Qidong Hu. PIWI-like proteins and piRNAs in breast cancer cell proliferation and migration. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A29.

The DExH box helicase domain of spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis.

Transposable selfish genetic elements have the potential to cause debilitating mutations as they replicate and reinsert within the genome. Therefore, it is critical to keep the cellular levels of these elements low. This is especially true in the germline where these mutations could affect the viability of the next generation. A class of small noncoding RNAs, the Piwi-associated RNAs, is responsible for silencing transposable elements in the germline of most organisms. Several proteins have been identified as playing essential roles in piRNA generation and transposon silencing. However, for the most part their function in piRNA generation is currently unknown. One of these proteins is the Drosophila melanogaster DExH box/Tudor domain protein Spindle-E, whose activity is necessary for the generation of most germline piRNAs. In this study we molecularly and phenotypically characterized 14 previously identified spindle-E alleles. Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain. Additionally, we found that processes that depend on piRNA function, including Aubergine localization, Dynein motor movement, and retrotransposon silencing, were severely disrupted in alleles with DExH box domain mutations. The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much. From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.

Getting a Grip on piRNA Cluster Transcription

The generation of piRNAs from long primary transcripts requires specialized factors that distinguish these precursors from canonical RNA polymerase II transcripts. Mohn et al. and Zhang et al. provide evidence that in Drosophila melanogaster noncanonical transcription coupled with splicing inhibition differentiates piRNA precursors from mRNAs and ensures their correct processing.

A C-Terminal Transmembrane Anchor Targets the Nuage-Localized Spermatogenic Protein Gasz to the Mitochondrial Surface.

Mitochondria, normally tubular and distributed throughout the cell, are instead found in spermatocytes in perinuclear clusters in close association with nuage, an amorphous organelle composed of RNA and RNA-processing proteins that generate piRNAs. piRNAs are a form of RNAi required for transposon suppression and ultimately fertility. MitoPLD, another protein required for piRNA production, is anchored to the mitochondrial surface, suggesting that the nuage, also known as intermitochondrial cement, needs to be juxtaposed there to bring MitoPLD into proximity with the remainder of the piRNA-generating machinery. However, the mechanism underlying the juxtaposition is unknown. Gasz, a multidomain protein of known function found in the nuage in vertebrates, is required for piRNA production and interacts with other nuage proteins involved in this pathway. Unexpectedly, we observed that Gasz, in nonspermatogenic mammalian cells lines, localizes to mitochondria and does so through a previously unrecognized conserved C-terminal mitochondrial targeting sequence. Moreover, in this setting, Gasz is able to recruit some of the normally nuage-localized proteins to the mitochondrial surface. Taken together, these findings suggest that Gasz is a nuage-localized protein in spermatocytes that facilitates anchoring of the nuage to the mitochondrial surface where piRNA generation takes place as a collaboration between nuage and mitochondrial-surface proteins.

Integrative Proteomic and Transcriptomic Analyses Reveal Multiple Post-transcriptional Regulatory Mechanisms of Mouse Spermatogenesis

Mammalian spermatogenesis consists of many cell types and biological processes and serves as an excellent model for studying gene regulation at transcriptional and post-transcriptional levels. Many key proteins, miRNAs, and perhaps piRNAs have been shown to be involved in post-transcriptional regulation of spermatogenesis. However, a systematic method for assessing the relationship between protein and mRNA expression has not been available for studying mechanisms of post-transcriptional regulation. In the present study, we used the iTRAQ-based quantitative proteomic approach to identify 2008 proteins in mouse type A spermatogonia, pachytene spermatocytes, round spermatids, and elongative spermatids with high confidence. Of these proteins, 1194 made up four dynamically changing clusters, which reflect the mitotic amplification, meiosis, and post-meiotic development of germ cells. We identified five major regulatory mechanisms termed "transcript only," "transcript degradation," "translation repression," "translation de-repression," and "protein degradation" based on changes in protein level relative to changes in mRNA level at the mitosis/meiosis transition and the meiosis/post-meiotic development transition. We found that post-transcriptional regulatory mechanisms are related to the generation of piRNAs and antisense transcripts. Our results provide a valuable inventory of proteins produced during mouse spermatogenesis and contribute to elucidating the mechanisms of the post-transcriptional regulation of gene expression in mammalian spermatogenesis.

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.

Structural insights into piRNA recognition by the human PIWI‐like 1 PAZ domain

Major advancements have been made in the past decade towards understanding the regulatory role of small non-coding RNAs (ncRNAs) in diverse development processes1. An integral part of this regulation is the conserved PIWI/Argonaute protein family. Phylogenetic analysis indicates that this protein family can be divided into two sub-families: the Piwi sub-family, named after Drosophila melanogaster protein PIWI (P-element induced wimpy testis), and the Ago sub-family, based on its founding member in Arabidopsis thaliana (Ago). Ago proteins are involved in RNA-mediated gene silencing complexes and utilize short guide RNAs to target specific nucleic acids1-4, whereas Piwi proteins bind to a novel class of ncRNAs called PIWI-interacting RNAs (piRNAs) that play diverse functions in germline development and gametogenesis5-11. Piwi/Ago proteins have a molecular weight of about 90 kDa and are defined by three protein motifs: the PAZ, MID and PIWI domains. The N-terminal PAZ domain is accountable for binding the 3′-end of the guide strand RNA; the middle MID domain is responsible for binding the 5′-phosphate RNA; and the following C-terminal PIWI domain has an RNase H endonuclease fold that is responsible for the RNA cleavage. The best-characterized small RNA partners of Ago proteins are microRNAs (miRNAs). Ago proteins utilize miRNAs to silence genes post-transcriptionally or use small-interfering RNAs (siRNAs) in both transcription and post-transcription silencing mechanisms12-17. However, Piwi proteins are restricted to germline cells and stem cells, and have been found to interact with a novel class of piRNAs (∼28-33 nucleotides) longer than miRNAs and siRNAs (∼20 nucleotides), suggesting novel functions distinct from those of Ago proteins18-22. piRNAs are 28 to 33 nucleotides in length and have been characterized by the cloning of small RNAs from anti-Piwi immunoprecipitates prepared from mammalian testes7-9. As the proportion of repeat elements able to generate piRNAs is actually smaller within the piRNA regions than the frequency of repeat sequences in the mouse genome, they are not believed to be derived from repeat sequences. It is believed that piRNAs are processed from single-stranded primary transcripts that are transcribed from defined genomic regions and have a preference for a uridine at their 5′ terminal6-10,23. Mammalian piRNAs are a highly complex mix of sequences, with tens of thousands of distinct piRNAs generated from the 50 to 100 defined primary transcripts. This may suggest that mammalian piRNAs, unlike miRNAs, are not processed in a precise manner. However, approximately 20% of all piRNA sequences were cloned three or more times, and many piRNA sequences from the same strand are partially overlapping, suggesting a quasi-random mechanism. The mechanism of biogenesis of D. melanogaster rasiRNAs is beginning to be elucidated, and may offer parallels for a specific mode of processing for piRNAs as well. piRNA biogenesis is thought to be Dicer-independent and they appear to be 2′-O'methylated at their 3′ terminal24-26. Between mammals, mature piRNAs are not conserved; however, the genomic regions, from which they derive, in particular the promoter sequences, are conserved. Mammalian piRNAs are strongly expressed in the male germline, their total number per cell obtained from testis tissue reaching up to two million – about 10-fold higher than the miRNA content of these cells. Despite their importance in developmental biology, a mechanistic understanding of the biogenesis of piRNAs is lacking, and the specific functional role PIWI proteins play in piRNA generation remains elusive. To understand the molecular basis of piRNA recognition by PIWI proteins, we have determined the three-dimensional solution structures of the human PIWI-like 1 (hPIWIL1) PAZ domain in its free form, as well as bound to a ssRNA (5′-pUGACA) oligonucleotide using nuclear magnetic resonance (NMR) spectroscopy methods.

PiRNA-Associated Germline Nuage Formation and Spermatogenesis Require MitoPLD Profusogenic Mitochondrial-Surface Lipid Signaling

The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD(-/-) mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates nuage components critical for piRNA production.

Transposon Silencing by piRNAs

In animal cells, small RNA molecules, called piRNAs, defend the genome against selfish DNA elements such as transposons. In this issue, Klattenhoff et al. (2009) report that an HP1 family protein, Rhino, is required for piRNA generation and transposon silencing in Drosophila germline cells. The results provide a link between heterochromatin and piRNA-mediated genome defense.

Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary

In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.