Ivan Olovnikov

Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons.

In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline.

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.

Euchromatic transposon insertions trigger production of novel Pi- and endo-siRNAs at the target sites in the drosophila germline.

The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA–mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3′ untranslated region of actively transcribed genes induces piRNA production towards the 3′-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.

Small RNA in the nucleus: the RNA-chromatin ping-pong

Eukaryotes use several classes of small RNA molecules to guide diverse protein machineries to target messenger RNA. The role of small RNA in post-transcriptional regulation of mRNA stability and translation is now well established. Small RNAs can also guide sequence-specific modification of chromatin structure and thus contribute to establishment and maintenance of distinct chromatin domains. In this review we summarize the model for the inter-dependent interaction between small RNA and chromatin that has emerged from studies on fission yeast and plants. We focus on recent results that link a distinct class of small RNAs, the piRNAs, to chromatin regulation in animals.

De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

Prokaryotic Argonautes defend genomes against invasive DNA

Argonaute proteins are central players in small RNA-mediated silencing mechanisms such as RNA interference (RNAi), microRNA repression and piRNA-mediated transposon silencing. In eukaryotes, Argonautes bind small RNAs that guide them to RNA targets in order to regulate gene expression and repress invasive genomic elements. Although Argonaute proteins are conserved in all life forms from bacteria to eukaryotes, until now studies have focused on the biological functions of eukaryotic Argonautes. Here we highlight two recent studies that discover the functions of prokaryotic Argonautes in defence against exogenous DNA.

Telomeric repeat silencing in germ cells is essential for early development in Drosophila

The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. We analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. Our data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

Natural variation of piRNA expression affects immunity to transposable elements

In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain.

Telomeric Retrotransposon HeT-A Contains a Bidirectional Promoter that Initiates Divergent Transcription of piRNA Precursors in Drosophila Germline

PIWI-interacting RNAs (piRNAs) provide the silencing of transposable elements in the germline. Drosophila telomeres are maintained by transpositions of specialized telomeric retroelements. piRNAs generated from sense and antisense transcripts of telomeric elements provide telomere length control in the germline. Previously, we have found that antisense transcription of the major telomeric retroelement HeT-A is initiated upstream of the HeT-A sense transcription start site. Here, we performed a deletion analysis of the HeT-A promoter and show that common regulatory elements are shared by sense and antisense promoters of HeT-A. Therefore, the HeT-A promoter is a bidirectional promoter capable of processive sense and antisense transcription. Ovarian small RNA data show that a solo HeT-A promoter within an euchromatic transgene initiates the divergent transcription of transgenic reporter genes and subsequent processing of these transcripts into piRNAs. These events lead to the formation of a divergent unistrand piRNA cluster at solo HeT-A promoters, in contrast to endogenous telomeres that represent strong dual-strand piRNA clusters. Solo HeT-A promoters are not immunoprecipitated with heterochromatin protein 1 (HP1) homolog Rhino, a marker of the dual-strand piRNA clusters, but are associated with HP1 itself, which provides piRNA-mediated transcriptional repression of the reporter genes. Unlike endogenous dual-strand piRNA clusters, the solo HeT-A promoter does not produce overlapping transcripts. In a telomeric context, however, bidirectional promoters of tandem HeT-A repeats provide a read-through transcription of both genomic strands, followed by Rhi binding. These data indicate that Drosophila telomeres share properties of unistrand and dual-strand piRNA clusters.

Interaction of Telomeric Retroelement HeT-A Transcripts and Their Protein Product Gag in Early Embryogenesis of Drosophila.

The telomere is a nucleoprotein complex at the ends of linear chromosomes that protects them from fusion and degradation. The telomere consists of telomeric DNA, a protective protein complex and telomeric RNA. Biogenesis of telomeric transcripts in development is still far from being understood. Drosophila telomeres are elongated by a transposition of specialized telomeric retrotransposons that encode proteins. Using transgenic constructs encoding tagged telomeric protein, we found that transcripts of Drosophila telomeric element HeT-A bind Gag-HeT-A protein encoded by these transcripts. Maternal HeT-A transcripts and Gag-HeT-A form ribonucleoprotein granules around centrosomes, centers of microtubule organization, during blastoderm formation, upon disruption of telomere silencing during oogenesis. The specific localization of HeT-A RNA is dependent on microtubules since disruption of microtubules caused delocalization of HeT-A transcripts. This transgenic system is a valuable model for the study of telomeric RNA biogenesis.