Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline. To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins. piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline.

Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) were discovered in different species, however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we show that the piRNA pathway provides a strong germline-specific mechanism of telomere homeostasis. We show that telomeric retrotransposon arrays belong to a unique class of dual-strand piRNA clusters whose transcripts, required for telomere elongation, serve simultaneously as piRNA precursors and their only targets. However, the ability to produce piRNAs and bind Rhino – a germline-specific homolog of heterochromatic protein 1 (HP1) – varies along telomeres. Most likely, this heterogeneity is determined by the peculiarities of telomeric retrotransposons themselves. piRNAs play a pivotal role in the establishment and maintenance of telomeric and subtelomeric chromatin in the germline facilitating loading of HP1 and histone 3 lysine 9 trimethylation mark – highly conservative telomere components – at different telomeric regions. piRNA pathway disruption results in telomere dysfunction characterized by a loss of heterochromatic components and translocation of telomeres from the periphery to the nuclear interior but does not affect the telomere end capping.