One Loop to Rule Them All: The Ping-Pong Cycle and piRNA-Guided Silencing.

The PIWI-interacting RNA (piRNA) pathway is a conserved defense mechanism that protects the genetic information of animal germ cells from the deleterious effects of molecular parasites, such as transposons. Discovered nearly a decade ago, this small RNA silencing system comprises PIWI-clade Argonaute proteins and their associated RNA-binding partners, the piRNAs. In this review, we highlight recent work that has advanced our understanding of how piRNAs preserve genome integrity across generations. We discuss the mechanism of piRNA biogenesis, give an overview of common themes as well as differences in piRNA-mediated silencing between species, and end by highlighting known and emerging functions of piRNAs

Rapid evolution of promoters from germline-specifically expressed genes including transposon silencing factors

Background: The piRNA pathway in animal gonads functions as an ‘RNA-based immune system’, serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23–28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways. Results: By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, we demonstrate that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. Our findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, we observe that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression. Conclusion: Our results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons

Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.We thank the Cold Spring Harbor Laboratory Microscopy Shared Resources for assistance, which are funded in part by Cancer Center Support Grant 5P30CA045508. This work was supported in part by a grant from the STARR Cancer Consortium, grants from the National Institutes of Health (NIH MERIT Award, R37GM062534 to G. J.H.), and a generous gift from Kathryn W. Davis to G.J.H. N.P. and G.J.H. are or were Investigators of the Howard Hughes Medical Institute. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Cell lines have been deposited by A.S. at the Drosophila Genomics Resource Center (NIH 2P40OD010949-10A1). G.J.H. is supported by Cancer Research UK and is a Wellcome Trust Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Press at http://dx.doi.org/10.1101/gad.284927.116

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production

piRNA-guided co-transcriptional silencing coopts nuclear export factors.

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing

Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis

The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 min and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells

A case-control study of apolipoprotein E genotypes in Alzheimer's disease associated with Down syndrome.

The prevalence of clinical signs and neuropathological findings of Alzheimer's disease (AD) is high in Down's syndrome (DS). In the general population, the apolipoprotein E (ApoE) epsilon 4 isoform is an important risk for AD. We studied the allelic frequencies of ApoE in 26 DS cases fulfilling clinical diagnostic criteria for AD and in 26 DS controls matched for age, sex, and premorbid level of mental retardation. A meta-analysis of data available in the literature was used for comparison with allele frequencies in other AD and control populations. ApoE type 2, 3, or 4 allele frequencies were not significantly different in AD-DS cases and DS controls. The ApoE epsilon 4 frequency in DS cases with AD (0.14; CI, 0.06-0.26) was significantly lower than in any other AD population studied so far and it is within the range of nondemented controls from the general population. These findings suggest that ApoE epsilon 4 does not significantly affect the pathogenesis of AD in DS patients

Production of artificial piRNAs in flies and mice

In animals a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. Presently, little is known about the signals that distinguish a locus as a source of piRNAs. It is also unknown how individual piRNAs are selected from long precursor transcripts. To address these questions, we inserted new artificial sequence information into piRNA clusters and introduced these marked clusters as transgenes into heterologous genomic positions in mice and flies. Profiling of piRNA from transgenic animals demonstrated that artificial sequences were incorporated into the piRNA repertoire. Transgenic piRNA clusters are functional in non-native genomic contexts in both mice and flies, indicating that the signals that define piRNA generative loci must lie within the clusters themselves rather than being implicit in their genomic position. Comparison of transgenic animals that carry insertions of the same artificial sequence into different ectopic piRNA-generating loci showed that both local and long-range sequence environments inform the generation of individual piRNAs from precursor transcripts

Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex