Paramutation in Drosophila Requires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and Induces Cis-spreading of piRNA Production

Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.     

Distinct functions for the Drosophila piRNA pathway in genome maintenance and telomere protection

Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. Here we show that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in lig-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex.     

Adaptation to P element transposon invasion in Drosophila melanogaster

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.     

piRNA dynamics in divergent zebrafish strains reveal long-lasting maternal influence on zygotic piRNA profiles

Transposable elements (TEs) are mobile genetic elements that can have many deleterious effects on the fitness of their host. The germline-specific PIWI pathway guards the genome against TEs, deriving its specificity from sequence complementarity between PIWI-bound small RNAs (piRNAs) and the TEs. The piRNAs are derived from so-called piRNA clusters. Recent studies have demonstrated that the piRNA repertoire can be adjusted to accommodate recent TE invasions by capturing invading TEs in piRNA loci. Thus far, no information concerning piRNA divergence is available from vertebrates. We present piRNA analyses of two relatively divergent zebrafish strains. We find that significant differences in the piRNA populations have accumulated, most notably among active class I TEs. This divergence can be split into differences in piRNA abundance per element and differences in sense/antisense polarity ratios. In crosses between animals of the different strains, many of these differences are resolved in the progeny. However, some differences remain, often leaning to the maternally contributed piRNA population. These differences can be detected at least two generations later. Our data illustrate, for the first time, the fluidity of piRNA populations in vertebrates and how the established diversity is transmitted to future generations.     

Structural insights into Rhino‐Deadlock complex for germline piRNA cluster specification

PIWI‐interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species‐specific manner and so defines the piRNA‐producing loci in the Drosophila genome. Here, we determine the crystal structures of Rhino‐Deadlock complex in Drosophila melanogaster and simulans. In both species, one Rhino binds the N‐terminal helix–hairpin–helix motif of one Deadlock protein through a novel interface formed by the beta‐sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. Our structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross‐species incompatibility between the sibling species. By determining the molecular architecture of this piRNA‐producing machinery, we discover a novel HP1‐partner interacting mode that is crucial to piRNA biogenesis and transposon silencing. We thus explain the cross‐species incompatibility of two sibling species at the molecular level.     

piRNA clusters as a main source of small RNAs in the animal germline

PIWI subfamily Argonaute proteins and small RNAs bound to them (PIWI interacting RNA, piRNA) control mobilization of transposable elements (TE) in the animal germline. piRNAs are generated by distinct genomic regions termed piRNA clusters. piRNA clusters are often extensive loci enriched in damaged fragments of TEs. New TE integration into piRNA clusters causes production of TE-specific piRNAs and repression of cognate sequences. piRNAs are thought to be generated from long single-stranded precursors encoded by piRNA clusters. Special chromatin structures might be essential to distinguish these genomic loci as a source for piRNAs. In this review, we present recent findings on the structural organization of piRNA clusters and piRNA biogenesis in Drosophila and other organisms, which are important for understanding a key epigenetic mechanism that provides defense against TE expansion.     

The piRNA pathway: a fly's perspective on the guardian of the genome

Throughout the eukaryotic lineage, small RNA silencing pathways protect the genome against the deleterious influence of selfish genetic elements such as transposons. In animals an elaborate small RNA pathway centered on PIWI proteins and their interacting piRNAs silences transposons within the germline. In contrast to other small RNA silencing pathways, we lack a mechanistic understanding of this genome defense system. However, genetic and molecular studies have uncovered a fascinating conceptual framework for this pathway that is conserved from sponges to mammals. We discuss our current understanding of the piRNA pathway in Drosophila with an emphasis on origin and biogenesis of piRNAs.     

A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice

piRNAs and Piwi proteins have been implicated in transposon control and are linked to transposon methylation in mammals. Here we examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells.     

A sensitive multiplex assay for piRNA expression

PIWI-interacting RNAs (piRNAs) are a new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins, Mili and Miwi. To help to decipher the mechanism of piRNA function, here, we report a real time PCR-based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with the Northern blot result. The method we developed can simultaneously detect at least eight piRNAs using only 10 pg total RNA, which is equivalent to the RNA present in a single cell. This is five to six order magnitude more sensitive than corresponding Northern blot assays. Finally we used this assay to analyze eight piRNAs expression in mouse primordial germ cells (PGCs) in genital ridges from E12.5, at the time when piRNA-binding protein Mili starts to be detected in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA from a single cell. This approach will help to understand the mechanism and function of piRNAs during germ cell development.