Sergey Shpiz

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.

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.

rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus

Telomeres in Drosophila are maintained by the specialized telomeric retrotransposons HeT-A, TART and TAHRE. Sense transcripts of telomeric retroelements were shown to be the targets of a specialized RNA-interference mechanism, a repeat-associated short interfering (rasi)RNA-mediated system. Antisense rasiRNAs play a key role in this mechanism, highlighting the importance of antisense expression in retrotransposon silencing. Previously, bidirectional transcription was reported for the telomeric element TART. Here, we show that HeT-A is also bidirectionally transcribed, and HeT-A antisense transcription in ovaries is regulated by a promoter localized within its 3′ untranslated region. A remarkable feature of noncoding HeT-A antisense transcripts is the presence of multiple introns. We demonstrate that sense and antisense HeT-A-specific rasiRNAs are present in the same tissue, indicating that transcripts of both directions may be considered as natural targets of the rasiRNA pathway. We found that the expression of antisense transcripts of telomeric elements is regulated by the RNA silencing machinery, suggesting rasiRNA-mediated interplay between sense and antisense transcripts in the cell. Finally, this regulation occurs in the nucleus since disruption of the rasiRNA pathway leads to an accumulation of TART and HeT-A transcripts in germ cell nuclei.

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.

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.

Role of piRNAs in the Drosophila telomere homeostasis.

Drosophila telomeres are maintained as a result of transpositions of specialized telomeric retrotransposons. The abundance of telomeric retroelement transcripts, as well as the frequency of their transpositions onto the chromosome ends, is controlled by a PIWI-interacting RNA (piRNA) pathway. In our recent report, we demonstrate strong evidence of piRNA-mediated transcriptional silencing of telomeric repeats in the Drosophila germline. Telomerase-generated repeats serve as a platform for recruiting specialized DNA-binding proteins which are involved in chromosome end protection and in the telomere length control. No specific proteins are known to bind to heterogeneous long sequences of the Drosophila telomeric retrotransposons. The importance of the piRNA silencing mechanism in the formation of telomeric chromatin along the region of the retrotransposon array will be discussed. We propose that Drosophila telomeric retrotransposon HeT-A serves as a template for the piRNA-mediated assembly of the specific protein complex, which is functionally similar to the recruiting of the DNA-binding telomeric proteins by the telomerase-generated repeats. The role of the piRNA pathway components in the assembly of the telomere capping complex was recently unveiled. Taken together, these data elucidate the importance of the piRNA pathway in the Drosophila telomere homeostasis.

Combined RNA/DNA Fluorescence In Situ Hybridization on Whole-Mount Drosophila Ovaries

DNA FISH (fluorescent in situ hybridization) analysis reveals the chromosomal location of the gene of interest. RNA in situ hybridization is used to examine the amounts and cell location of transcripts. This method is commonly used to describe the localization of processed transcripts in different tissues or cell lines. Gene activation studies are often aimed at determining the mechanism of this activation (transcriptional or posttranscriptional). Elucidation of the mechanism of piRNA-mediated silencing of genomic repeats is at the cutting edge of small RNA research. The RNA/DNA FISH technique is a powerful method for assessing transcriptional changes at any particular genomic locus. Colocalization of the RNA and DNA FISH signals allows a determination of the accumulation of nascent transcripts at the transcribed genomic locus. This would be suggest that this gene is activated at the transcriptional (or co-transcriptional) level. Moreover, this method allows for the identification of transcriptional derepression of a distinct copy (copies) among a genomic repeat family. Here, a RNA/DNA FISH protocol is presented for the simultaneous detection of RNA and DNA in situ on whole-mount Drosophila ovaries using tyramide signal amplification. With subsequent immunostaining of chromatin components, this protocol can be easily extended for studying the interdependence between chromatin changes at genomic loci and their transcriptional activity.

Analyses of piRNA-Mediated Transcriptional Transposon Silencing in Drosophila : Nuclear Run-On Assay on Ovaries

In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. piRNA pathway mutations lead to overexpression and mobilization of retrotransposons in the germline. In different organisms, small RNAs were shown to be implicated in the posttranscriptional degradation of mRNA and/or transcriptional repression of the homologous locus. In Drosophila, the mechanism of piRNA-mediated silencing is still far from being understood. Transcriptional silencing implies a piRNA-mediated formation of repressive chromatin which diminishes the transcriptional capacity of the target locus. Nuclear Run-On (NRO) assay allows a direct estimation of the density of transcribing polymerases at specific genomic regions. Here we describe the NRO protocol on Drosophila ovarian tissues which can be useful for investigation of the transcriptional silencing in the female germline.

Control of Telomere Length in Drosophila

The problem of incomplete end replication of DNA was originally raised by the Russian scientist Aleksey Olovnikov (Olovnikov, 1971, 1973). The main function of telomeric DNA is the compensation of end degradation. In most eukaryotes, telomeric DNA is maintained by the action of telomerase, which is responsible for the synthesis of short 6-9 nucleotide repeats using an RNA component as a template (Greider and Blackburn, 1985). In contrast, telomeres of Drosophila are maintained as a result of retrotransposition of specialized telomeric non-long terminal repeat retrotransposons (Biessmann et al., 1990a; Biessmann et al., 1992a; Levis et al., 1993; Abad et al., 2004b).