piRNA Expression Research Articles

Silencing of LINE-1 retrotransposons contributes to variation in small noncoding RNA expression in human cancer cells.

Noncoding RNAs are key players in the maintenance of genomic integrity, particularly in silencing the expression of repetitive elements, some of which are retrotransposable and capable of causing genomic instability. Recent computational studies suggest an association between L1 expression and the generation of small RNAs. However, whether L1 expression has a role in the activation of small RNA expression has yet to be determined experimentally. Here we report a global analysis of small RNAs in deep sequencing from L1-active and L1-silenced breast cancer cells. We found that cells in which L1 expression was silenced exhibited greatly increased expression of a number of miRNAs and in particular, members of the let-7 family. In addition, we found differential expression of a few piRNAs that might potentially regulate gene expression. We also report the identification of several repeat RNAs against LTRs, LINEs and SINE elements. Although most of the repeat RNAs mapped to L1 elements, in general we found no significant differences in the expression levels of repeat RNAs in the presence or absence of L1 expression except for a few RNAs targeting subclasses of L1 elements. These differentially expressed small RNAs may function in human genome defence responses.

Elevated expression of Piwi and piRNAs in ovaries of triploid crucian carp

Increasing piRNAs provide RNA-interference pathways to regulate transposons and gene expression in germline cells. We demonstrate that Piwi transcripts are exclusively expressed in adult testes and ovaries in teleosts, with triploids showing the highest Piwi expression in the ovaries. Studies in vivo and in vitro showed that hCG and E2 treatment suppressed Piwi expression. We further cloned 200 small RNAs in the three kinds of fish. Seven piRNAs were obtained from all the three different ploidy fishes. During ovulation, five piRNAs showed significantly higher expression in the ovaries of sterile triploids than fertile diploids and tetraploids. Furthermore, E2 suppressed the expression of the six piRNAs at different levels in vivo and in vitro. The present study bridges the gap between the HPG axis and Piwi–piRNA pathway by suggesting that a dysfunctional HPG axis abrogated the piRNA suppression in triploid fish.

Small RNA regulation of reproductive function

Post-transcriptional gene regulation is one mechanism that occurs "above the genome," allowing the cells of an organism to have dramatically different phenotypes and functions. Non-coding ribonucleic acid (ncRNA) molecules regulate transcript and protein abundance above the level of transcription, and appear to play substantial roles in regulation of reproductive tissues. Three primary classes of small ncRNA are microRNA (miRNA), endogenous small interfering RNA (endo-siRNA), and PIWI-interacting RNA (piRNA). These RNA classes have similarities and clear distinctions between their biogenesis and in the interacting protein machinery that facilitate their effects on cellular phenotype. Characterization of the expression and importance of the critical components for the biogenesis of each class in different tissues is continuously contributing a better understanding of each of these RNA classes in different reproductive cell types. Here, we discuss the expression and potential roles of miRNA, endo-siRNA, and piRNA in reproduction from germ-cell development to pregnancy establishment and placental function. Additionally, the potential contribution of RNA binding proteins, long ncRNAs, and the more recently discovered circular RNAs (circRNAs) in relation to small RNA function is discussed.

Knockdown Of The Piwi - Like Protein 4 (PIWIL4) Delays Leukemic Growth and Is Associated With Gross Changes In The Global Histone Methylation Marks In Human MLL - Rearranged AML

Piwi proteins belong to a class of proteins which were shown to be critically involved in the maintenance of the self-renewal property of stem cells in lower organisms. Furthermore, it was shown that they preserve genomic integrity through epigenetic silencing of transposable elements via CpG methylation and repressive histone modifications such as H3K9me3 in close interaction with a novel class of non-coding RNA called piRNA. So far there are neither precise data on the function of Piwi proteins in human acute myeloid leukemia, nor are there reports on expression of piRNAs in this disease. In a first step we tested PIWIL gene expression levels in normal human hematopoietic cells and leukemic patient samples by qRT-PCR. Among the family of human PIWI genes, PIWIL4 showed the highest expression level and was ubiquitously expressed in normal hematopoietic stem/progenitors, mature lymphoid and myeloid cells. Importantly, PIWIL4 showed aberrantly high expression in more than 72% of the AML patients (n=68; p< 0.0001) compared to normal CD34+ bone marrow (BM) and total BM cells (n=3). Notably, in nine of the ten MLL-AF9 rearranged AML patients, PIWIL4 was 64-fold higher expressed compared to normal CD34+ BM (p<0.0001) and 8-fold higher compared to inv(16), PML-RARa or cytogenetically normal AML patients (p<0.0001). To further validate this finding we analysed gene expression data performed on CD34+ human cord blood cells transduced with MLL-AF9 (n=9) vs AML-ETO (n=6) vs MYH11 (n=3): of note, PIWIL4 showed a 6 fold increase in expression in the MLL-AF9 transduced cells compared to the other experimental arms. Stable knockdown of PIWIL4 in the MLL rearranged AML cell lines MV4-11 (MLL-AF4) and THP-1 (MLL-AF9) significantly impaired growth in vitro (n=3) reducing proliferation and clonogenic growth by 83%/93% and 91%/93%, respectively. In addition, depletion of PIWIL4 delayed onset of leukemia in NSG mice transplanted with MV4-11/ THP-1 cells transduced with shPIWIL4 compared to the scrambled control (shRNA: AML onset 48/62d after transplantation vs. 30/30 days in the scrambled control; n=4/8 per arm; p< 0.0001/p<0.001). ChIP-seq analysis revealed that depletion of PIWIL4 in the THP1 cell line results in a marked global reduction in repressive H3K9me3 marks and in an increase in activating H3K4me3 marks as compared to cells transduced with the scrambled control. RNA-seq analyses revealed over 2500 differentially expressed genes upon PIWIL4 depletion with 60% of the genes being upregulated compared to the scrambled control (p<0.05). Among them genes involved in cell cycle such as RB1, P21, TGFB1 as well as epigenetic modifiers such as SETDB1, HDAC1,2 and demethylating enzyme TDG were differentially expressed. RB1 and EED, a protein necessary for PRC2 complex function, displayed an increase in expression and loss of H3K9me3 modifications on their promoters upon knockdown of PIWIL4. To prove piRNA expression in human AML and to test any association between PIWIL4 expression and piRNA signatures, microarray analyses covering 23,677 piRNAs was performed on the MLL-AF9 rearranged THP-1 cell line, of which 14193 piRNAs showed expression levels higher than 4 (arbitrary log2 scale). PIWIL4 knockdown induced differential expression of 981 piRNAs (p≤0.01, fold change ≥2), of which 527 were downregulated and 454 upregulated. Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML, affects leukemic growth, shapes epigenetic marks and impacts piRNA expression in this disease. Disclosures:No relevant conflicts of interest to declare.

The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles

Mobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.

Prediction of transcribed PIWI-interacting RNAs from CHO RNAseq data

Chinese hamster ovary (CHO) cells are currently the most important mammalian host for the manufacture of biopharmaceuticals. To enhance our understanding of cellular processes, pathways, and the genetic setup of CHO cell lines, we predicted PIWI interacting RNAs (piRNAs) from small RNA sequencing data. Although piRNAs are the least understood class of small non-coding RNAs that mediate RNA silencing, it is believed that they play a pivotal role in protecting genome integrity by repressing transposable elements. Since genomic integrity is the key to prolonged stability of recombinant CHO cell lines, we characterized piRNA sequences and expression in six CHO cell lines by computational analysis of an existing small RNA sequencing dataset using proTRAC and the published CHO genome as reference. Here we present the result of this analysis consisting of 25,626 piRNAs and 540 piRNA clusters. Moreover we provide first evidence for differential piRNA expression in adherent and suspension-adapted CHO-K1 and DUKXB11 host cell lines as well as their recombinant derivatives, indicating that piRNAs might be tools for cell line development and engineering.

Defining piRNA expression

Defining piRNA expression

Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state

In the metazoan germline, piwi proteins and associated piwi-interacting RNAs (piRNAs) provide a defense system against the expression of transposable elements. In the cytoplasm, piRNA sequences guide piwi complexes to destroy complementary transposon transcripts by endonucleolytic cleavage. However, some piwi family members are nuclear, raising the possibility of alternative pathways for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromosomes. 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. Our 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.

Altered expression of piRNAs and their relation with clinicopathologic features of breast cancer

PIWI interacting RNAs (piRNAs) are endogenous regulatory non-coding RNAs which are generally expressed in the germline across animal species. The expressions of piRNAs in tumor tissue were rarely reported until now. The aim of this study was to investigate the expression of piRNAs in breast cancer. Deep sequencing was carried out in four tumor and four matched non-tumor tissues from resected breast cancer cases to screen out differentially expressed piRNAs. Furthermore, the result was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) technology in 50 patients with breast cancer and the piRNA expression levels were correlated with clinicopathologic features. Through deep sequencing, six differentially expressed piRNAs were screened out (5 up- and 1 down-regulated) in the four tumor tissues compared with matched non-tumor tissues. Among them, 4 piRNAs (piR-4987, piR-20365, piR-20485 and piR-20582) were confirmed to be up-regulated by realtime RT-PCR in 50 breast cancer (P < 0.001). Moreover, up-regulated piR-4987 expression was associated with lymph node positivity (P = 0.001). PiRNAs might play a role in breast cancer and act as tumor markers.

Altered Expression of Porcine Piwi Genes and piRNA during Development

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2–fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes.

PiRNA-regulated memory?

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs that have an unclear function. piRNA expression was thought to be limited to germline cells, but in a new study, Kandel and colleagues have detected piRNAs in a microRNA library from the CNS of Aplysia californica. Experiments involving knockdown of Piwi in cultured A. californica neurons revealed that Piwi–piRNA complexes enhance serotonin-induced long-term facilitation — a form of synaptic plasticity implicated in long-term memory. These complexes exerted their effects through methylation of the promoter region of the gene encoding CREB2, a major transcriptional repressor of memory formation in A. californica, leading to a decrease in CREB2 expression.

Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation

BackgroundIn the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet.ResultsHere we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries.ConclusionsCollectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype.

Pinpointing the expression of piRNAs and function of the PIWI protein subfamily during spermatogenesis in the mouse

PIWI proteins and piRNAs have been linked to transposon silencing in the primordial mouse testis, but their function in the adult testis remains elusive. Here we report the cytological characterization of piRNAs in the adult mouse testis and the phenotypic analysis of Miwi−/−; Mili−/− mice. We show that piRNAs are specifically present in germ cells, especially abundant in spermatocytes and early round spermatids, regardless of the type of the genomic sequences to which they correspond. piRNAs and PIWI proteins are present in both the cytoplasm and nucleus. In the cytoplasm, they are enriched in the chromatoid body; whereas in the nucleus they are enriched in the dense body, a male-specific organelle associated with synapsis and the formation of the XY body during meiosis. Moreover, by generating Miwi−/−; Mili−/− mice, which lack all PIWI proteins in the adult, we show that PIWI proteins and presumably piRNAs in the adult are required only for spermatogenesis. Spermatocytes without PIWI proteins are arrested at the pachytene stage, when the sex chromosomes undergo transcriptional silencing to form the XY body. These results pinpoint a function of the PIWI protein subfamily to meiosis during spermatogenesis.

MVH in piRNA processing and gene silencing of retrotransposons

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.

Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi

Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for germ cell development, but analysis of the molecular mechanisms of these ribonucleoproteins remains challenging in most animal germ cells. To address this challenge, we systematically characterized Xiwi, a Xenopus Piwi homologue, and piRNAs from Xenopus eggs and oocytes. We used the large size of Xenopus eggs to analyze small RNAs at the single cell level, and find abundant piRNAs and large piRNA clusters in the Xenopus tropicalis genome, some of which resemble the Drosophila piRNA-generating flamenco locus. Although most piRNA clusters are expressed simultaneously in an egg, individual frogs show distinct profiles of cluster expression. Xiwi is associated with microtubules and the meiotic spindle, and is localized to the germ plasm--a cytoplasmic determinant of germ cell formation. Xiwi associates with translational regulators in an RNA-dependent manner, but Xenopus tudor interacts with Xiwi independently of RNA. Our study adds insight to piRNA transcription regulation by showing that individual animals can have differential piRNA expression profiles. We suggest that in addition to regulating transposable elements, Xiwi may function in specifying RNA localization in vertebrate oocytes.

High-resolution profiling and discovery of planarian small RNAs

Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.

Requirement for Sun1 in the expression of meiotic reproductive genes and piRNA

The inner nuclear envelope (NE) proteins interact with the nuclear lamina and participate in the architectural compartmentalization of chromosomes. The association of NE proteins with DNA contributes to the spatial rearrangement of chromosomes and their gene expression. Sun1 is an inner nuclear membrane (INM) protein that locates to telomeres and anchors chromosome movement in the prophase of meiosis. Here, we have created Sun1-/- mice and have found that these mice are born and grow normally but are reproductively infertile. Detailed molecular analyses showed that Sun1-/- P14 testes are repressed for the expression of reproductive genes and have no detectable piRNA. These findings raise a heretofore unrecognized role of Sun1 in the selective gene expression of coding and non-coding RNAs needed for gametogenesis.

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.

The PIWI proteins SMEDWI-2 and SMEDWI-3 are required for stem cell function and piRNA expression in planarians

PIWI proteins are expressed in germ cells in a wide variety of metazoans, where they participate in the synthesis and function of a novel class of small RNAs called PIWI associated RNAs (piRNAs). One function of piRNAs is to preserve the integrity of the germline genome by silencing transposons, though they also participate in epigenetic and post-transcriptional gene regulation. In the planarian Schmidtea mediterranea, the PIWI proteins SMEDWI-1 and SMEDWI-2 are expressed in neoblasts and SMEDWI-2 is required for regeneration and homeostasis. Here, we identify a diverse population of approximately 32-nucleotide small RNAs that strongly resemble vertebrate and insect piRNAs and map to hundreds of thousands of loci in the S. mediterranea genome. The expression of these RNAs occurs predominantly in neoblasts and is not restricted to the germline. RNAi knockdown of either SMEDWI-2 or a newly identified PIWI protein, SMEDWI-3, impairs regeneration and homeostasis and decreases the levels of both piRNAs and neoblasts. Therefore, SMEDWI-2 and SMEDWI-3 are required for piRNA expression, regeneration, and neoblast function in S. mediterranea.

DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes

Silencing of transposable elements occurs during fetal gametogenesis in males via de novo DNA methylation of their regulatory regions. The loss of MILI (miwi-like) and MIWI2 (mouse piwi 2), two mouse homologs of Drosophila Piwi, activates retrotransposon gene expression by impairing DNA methylation in the regulatory regions of the retrotransposons. However, as it is unclear whether the defective DNA methylation in the mutants is due to the impairment of de novo DNA methylation, we analyze DNA methylation and Piwi-interacting small RNA (piRNA) expression in wild-type, MILI-null, and MIWI2-null male fetal germ cells. We reveal that defective DNA methylation of the regulatory regions of the Line-1 (long interspersed nuclear elements) and IAP (intracisternal A particle) retrotransposons in the MILI-null and MIWI2-null male germ cells takes place at the level of de novo methylation. Comprehensive analysis shows that the piRNAs of fetal germ cells are distinct from those previously identified in neonatal and adult germ cells. The expression of piRNAs is reduced under MILI- and MIWI2-null conditions in fetal germ cells, although the extent of the reduction differs significantly between the two mutants. Our data strongly suggest that MILI and MIWI2 play essential roles in establishing de novo DNA methylation of retrotransposons in fetal male germ cells.