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The RNA fluorescence in situ hybridization (FISH) technique combined with immunostaining is a powerful method to visualize a specific transcript and a protein of interest simultaneously. Although whole-mount RNA FISH is routinely used to determine RNA intracellular localization, a detailed picture of RNA distribution in complex tissues remains a challenge. The main problem is the various permeability of morphologically different cells within a tissue. We overcome this challenge by developing an approach based on differential permeabilization treatment of tissue specimens. We have tested and optimized conditions for RNA FISH combined with immunofluorescent staining (RNA FISH/IF) to detect the maternal telomeric retrotransposon HeT-A RNPs in the Drosophila ovaries and syncytial embryos. Methods described here are applicable to a broad variety of biological tissue specimens.

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction.

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.

Ccr4-Not is a highly conserved complex involved in cotranscriptional RNA surveillance pathways in yeast. In Drosophila, Ccr4-Not is linked to the translational repression of miRNA targets and the posttranscriptional control of maternal mRNAs during oogenesis and embryonic development. Here, we describe a new role for the Ccr4-Not complex in nuclear RNA metabolism in the Drosophila germline. Ccr4 depletion results in the accumulation of transposable and telomeric repeat transcripts in the fraction of chromatin-associated RNA; however, it does not affect small RNA levels or the heterochromatin state of the target loci. Nuclear targets of Ccr4 mainly comprise active full-length transposable elements (TEs) and telomeric and subtelomeric repeats. Moreover, Ccr4-Not foci localize at telomeres in a Piwi-dependent manner, suggesting a functional relationship between these pathways. Indeed, we detected interactions between the components of the Ccr4-Not complex and piRNA machinery, which indicates that these pathways cooperate in the nucleus to recognize and degrade TE transcripts at transcription sites. These data reveal a new layer of transposon control in the germline, which is critical for the maintenance of genome integrity.

Expression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a heterochromatin protein 1 (HP1) homolog. Previously, we have shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. We noted that identical transgenes located in different genomic sites vary considerably in piRNA production and classified them as “strong” and “weak” piRNA clusters. Here, we investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. We found that the formation of a transgenic piRNA cluster is accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the “empty” sites before transgene insertion. Finally, we have shown that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an “all-or-nothing” model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production.

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.

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.