M. S. Klenov

Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline

Proteins of the Argonaute family have been identified as key components of RNA interference (RNAi) pathway. RNAi-related mechanisms are implicated in the regulation of gene expression and repression of transposable elements in eukaryotes. The piwi gene encoding protein of the Drosophila Argonaute family was shown to be required for the germ stem cells maintenance. Here, we show that piwi is involved in silencing of LTR retrotransposons in testes. piwi mutations led to derepression of endogenous retrotransposon copia as well as to upregulation of the reporter gene driven by copia LTR. piwi mutation causes accumulation of retrotransposon mdg1 transcripts at the apical tip of testes, including germinal proliferative center where PIWI protein was shown to be expressed. We applied inverse PCR approach to detect the newly arisen insertions of the mdg1 retrotransposon in the progeny of individual piwi mutant males. Owing to piwi mutation a high rate of mdg1 transpositions was revealed. Thus, piwi is involved in the silencing of retrotransposons in the precursors of male gametes. Our results provide the first evidence that protein of the Argonaute family prevents retrotranspositions. It is supposed that the disturbance of RNA silencing system in germinal cells might cause transposition burst.

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.

Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line.

ABSTRACT To date, few natural cases of RNA-silencing-mediated regulation have been described. Here, we analyzed repression of testis-expressed Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that produce sense and antisense short RNAs. The Stellate promoter is dispensable for suppression, but local disturbance of complementarity between the Stellate transcript and the Su(Ste) repeats impairs silencing. Using in situ RNA hybridization, we found temporal control of the expression and spatial distribution of sense and antisense Stellate and Su(Ste) transcripts in germinal cells. Antisense Su(Ste) transcripts accumulate in the nuclei of early spermatocytes before the appearance of sense transcripts. The sense and antisense transcripts are colocalized in the nuclei of mature spermatocytes, placing the initial step of silencing in the nucleus and suggesting formation of double-stranded RNA. Mutations in the aubergine and spindle-E genes, members of the Argonaute and RNA helicase gene families, respectively, impair silencing by eliminating the short Su(Ste) RNA, but have no effect on microRNA production. Thus, different small RNA-containing complexes operate in the male germ line.

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.

Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster

Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation.

Heterochromatin formation: role of short RNAs and DNA methylation.

The role of small double-stranded RNAs is considered in formation of silent chromatin structure. Small RNAs are implicated in the regulation of individual gene transcription, suppression of transposon expression, and in maintaining functional structure of extended heterochromatic regions. Interrelations between short RNA-dependent gene silencing, histone modifications, and DNA methylation are discussed. Specific features of RNA-induced chromatin repression in various eucaryotes are also described.

Stellate repeats: targets of silencing and modules causing cis-inactivation and trans-activation.

The mechanism of silencing of testis expressed X-linked Stellate repeats by homologous Y-linked Suppressor of Stellate [Su(Ste)] repeats localized in the crystal locus was studied. The double stranded RNA as a product of symmetrical transcription of Su(Ste) repeat and small iinterfaceSu(Ste) siRNA were revealed suggesting the mechanism of RNA interference (RNAi) for Stellate silencing. The relief of Stellate silencing as a result of impaired complementarity between the sequences of putative target Stellate transcripts and Su(Ste) repeats was shown. The role of RNAi mechanism in the silencing of heterochromatic retrotransposon GATE inserted in Stellate cluster was revealed. The studies of cis-effects of Stellate tandem repeats causing variegated expression of juxtaposed reporter genes were extended and the lacZ variegation in imaginal disc was shown. The exceptional case of a non-variegated expression of mini-white gene juxtaposed to Stellate repeats in a construct inserted into the 39C region was shown to be accompanied by trans-activation in homozygous state. Trans-activation effect was retained after transposition of this construct into heterochromatic environment in spite of strong variegation of a mini-white gene.

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.