Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwi Nt , removing the nuclear localization signal of the Piwi protein. piwi Nt females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwi Nt mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwi Nt ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC selfrenewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells.oogenesis | short RNA

The Piwi-interacting RNA (piRNA)-interacting Piwi protein is involved in transcriptional silencing of transposable elements in ovaries of Drosophila melanogaster. Here we characterized the genome-wide effect of nuclear Piwi elimination on the presence of the heterochromatic H3K9me3 mark and HP1a, as well as on the transcription-associated mark H3K4me2. Our results demonstrate that a significant increase in the H3K4me2 level upon nuclear Piwi loss is not accompanied by the alterations in H3K9me3 and HP1a levels for several germline-expressed transposons, suggesting that in this case Piwi prevents transcription by a mechanism distinct from H3K9 methylation. We found that the targets of Piwi-dependent chromatin repression are mainly related to the elements that display a higher level of H3K4me2 modification in the absence of silencing, i.e. most actively transcribed elements. We also show that Piwi-guided silencing does not significantly influence the chromatin state of dual-strand piRNA-producing clusters. In addition, host protein-coding gene expression is essentially not affected due to the nuclear Piwi elimination, but we noted an increase in small nuclear spliceosomal RNAs abundance and propose Piwi involvement in their post-transcriptional regulation. Our work reveals new aspects of transposon silencing in Drosophila, indicating that transcription of transposons can underpin their Piwi dependent silencing, while canonical heterochromatin marks are not obligatory for their repression.

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.

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The evolutionarily conserved nuclear Piwi protein of Drosophila melanogaster is the defining member of the Argonaute small RNA binding protein family. Guided by piRNAs, Piwi functions in transpo son silencing in somatic and germ cells of gonads. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi accumulates predominantly in the nucleolus, the main nuclear compartment which participates not only in rRNA synthesis, but also in various cell stress responses. We have shown the colocalization of Piwi with the nucleolar marker proteins fibrillarin and Nopp140. The piwi Nt mutation which prevents the transport of Piwi to the nucleus and disrupts transposon silencing led to 6-8 fold upregulation of rRNA gene expression, as assessed by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depleted Piwi from the nucleolus. The same effect was observed upon inhibition of the activity of RNA polymerase I, which transcribes rRNA genes, but not RNA polymerase II. In contrast, upon heat shock, Piwi concentrated in the nucleolus and was depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase I activity modulation and stress response in the nucleolus. Possible noncanonical Piwi functions are discussed, which are not related to the role of Piwi in transposon silencing.