A. D. Stolyarenko

Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline

Silencing of genomic repeats, including transposable elements, in Drosophila melanogaster is mediated by repeat-associated short interfering RNAs (rasiRNAs) interacting with proteins of the Piwi subfamily. rasiRNA-based silencing is thought to be mechanistically distinct from both the RNA interference and microRNA pathways. We show that the amount of rasiRNAs of a wide range of retroelements is drastically reduced in ovaries and testes of flies carrying a mutation in the spn-E gene. To address the mechanism of rasiRNA-dependent silencing of retrotransposons, we monitored their chromatin state in ovaries and somatic tissues. This revealed that the spn-E mutation causes chromatin opening of retroelements in ovaries, resulting in an increase in histone H3 K4 dimethylation and a decrease in histone H3 K9 di/trimethylation. The strongest chromatin changes have been detected for telomeric HeT-A elements that correlates with the most dramatic increase of their transcript level, compared to other mobile elements. The spn-E mutation also causes depletion of HP1 content in the chromatin of transposable elements, especially along HeT-A arrays. We also show that mutations in the genes controlling the rasiRNA pathway cause no derepression of the same retrotransposons in somatic tissues. Our results provide evidence that germinal Piwi-associated short RNAs induce chromatin modifications of their targets.

The RNA Interference Proteins and Vasa Locus are Involved in the Silencing of Retrotransposons in the Female Germline of Drosophila melanogaster

RNA interference (RNAi) is considered as a defense against expansion of transposable elements. The proteins related to RNA helicase and Argonaute families are involved in RNAi process in different organisms. It was shown that Argonaute AUBERGINE and putative RNA helicase SPINDLE-E proteins were essential for RNAi in Drosophila. Here, we describe the role of aubergine (aub) and spindle-E (spn-E) genes in the control of LTR retrotransposon copia and non-LTR telomeric Het-A and I retrotransposons in ovaries. spn-E mutation causes a drastically increased lacZ expression driven by copia LTR. For the first time we show the involvement of AUBERGINE protein and VASA RNA helicase, essential for oocyte patterning, in the retrotransposon silencing. spn-E, vasa and aub mutations cause similar accumulation of both I element and Het-A transcripts in the developing oocyte. VASA and AUBERGINE proteins are known as components of perinuclear ribonucleoprotein particles in germ cells, and spn-E mutation disturbs protein content of the particles. We suggest participation of these proteins in the same silencing pathway.

Separation of stem cell maintenance and transposon silencing functions of Piwi protein

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, piwiNt, removing the nuclear localization signal of the Piwi protein. piwiNt 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. piwiNt mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwiNt 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 self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells.

Impact of nuclear Piwi elimination on chromatin state in Drosophila melanogaster ovaries

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.

A novel organelle, the piNG-body, in the nuage of Drosophila male germ cells is associated with piRNA-mediated gene silencing

A novel perinuclear nuage organelle, the piNG-body, is associated with piRNA silencing in testes of Drosophila. This body contains the known ovarian nuage proteins Vasa, Aub, AGO3, Tud, Spn-E, Bel, Squ, and Cuff, as well as AGO1.

Role of short RNAs in regulating the expression of genes and mobile elements in germ cells

Two main types of short RNAs, 21 to 25 nucleotides long, are involved in the negative regulation of gene expression in eukaryotes: microRNAs and small interfering RNAs (siRNAs) of the RNA interference system. MicroRNAs predominantly suppress the translation of mRNA targets, while siRNAs not only prevent mRNA translation and/or lead to mRNA degradation, but are also involved in the regulation of gene expression at the transcriptional level. In germ cells, translational regulation of gene expression plays a significant role and its mechanism has been extensively studied in oogenesis of Drosophila. The role of heterochromatization and chromatin compaction, which can repress the expression of mobile elements and other repeated elements of the genome, was studied to a lesser extent. Activation and transposition of mobile elements accompanied by mutations and chromosome rearrangements are especially dangerous in germline cells. It has been proposed that a specialized class of short RNAs, repeat associated siRNAs (rasiRNAs), can be involved in repression of the expression of mobile elements in Drosophila germ cells. Here we describe the findings on subcellular ribonucleoprotein structures characteristic of germ cells: perinuclear and polar granules containing proteins of the RNA interference and microRNA maturation system. Also, we present our own results revealing the role of genes of the RNA interference system in mobile element silencing in Drosophila.

Piwi protein as a nucleolus visitor in Drosophila melanogaster

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 transposon 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 piwiNt 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.

Induction of transposon silencing in the Drosophila germline

In this review we consider the role of the piRNA system in transposable element silencing in the Drosophila melanogaster germline. We focus on new data that demonstrate the mechanisms of initiation of piRNA biogenesis in ovarian germinal cells and the role of Piwi protein in this process, including our own results.

Interplay of transposon-silencing genes in the germline of Drosophila melanogaster

Complexes of Piwi family proteins with short piRNAs (Piwi-interacting RNAs) are responsible for silencing transposable elements in animal reproductive organs. In Drosophila melanogaster, three proteins (Piwi, Aub, and Ago3) are members of the Piwi family. Piwi is the nuclear protein of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins involved in piRNA amplification in perinuclear granules that constitute special organelles of germinal cells called nuage. Mutations in genes of the piRNA system are known to cause derepression of several transposable elements. In this study, we compared quantitatively changes in expression of a larger number of elements in the case of mutations in the piwi gene, genes aub, mael, and spn-E, which encode proteins of nuage granules, and armi gene coding an RNA helicase, the lack of which does not interfere with cytoplasmic piRNA amplification but disturbs nuclear localization of Piwi protein. We found that the genes piwi, armi, aub, spn-E, and mael interact to induce silencing of some retrotransposons (HMS-Beagle, Gate and HeT-A); the same genes, except piwi, are involved in repression of I and G elements. We propose that Armi is involved in control of not only nuclear Piwi localization. Our data suggest the relation of nuage proteins Aub, Spn-E, and Mael to Piwi-mediated silencing of retrotransposons Gate and HMS-Beagle in the nucleus. In general, our results corroborate the idea of genome stabilization by means of various silencing strategies specific to different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in germinal cells.

Functions of piRNAs and the Piwi protein in Drosophila

Short regulatory RNAs 25–35 nucleotides in length, along with RNA-binding proteins of the Piwi family, constitute an evolutionarily conserved system that functions mainly in eukaryotic gonads. The system can be regarded as a variation of the RNA interference mechanism, which is based on the recognition of target RNA as a result of complementary interactions with piRNAs. Different variants of this regulatory system function both in germline cells, including stem cells, and somatic cells of the niche, ensuring maintenance of the germline stem cells and their differentiation. One of the most important functions (but not the only one) of this system is the repression of transposons, which guarantees genome stability in germline cells. This review focuses on the works of the authors in the context of outstanding international achievements in the rapidly evolving research area, the biology of piRNA and the functions of the Piwi protein.