Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline. Drosophila melanogaster

Background. 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. Results 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. Conclusions 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. Overall design: The fractions of small RNAs (19-29 nt) from ovaries of four transgenic strains of Drosophila melanogaster were sequenced using Illumina HiSeq 2000.

背景 与PIWI相互作用RNA（PIWI-interacting RNAs，piRNAs）相关的端粒小RNA已在多种真核生物中被报道，但其在生殖系特异性端粒功能中的作用仍知之甚少。本研究以果蝇（Drosophila）为模型，深入探究了端粒piRNA的生物发生过程及其在生殖系端粒稳态中的功能。结果 为全面表征端粒piRNA簇，我们整合了对内源端粒重复序列的分析数据，以及插入至不同端粒及亚端粒区域的转基因相关数据。携带端粒转基因的菌株的小RNA测序数据显示，所有转基因均属于双链piRNA簇类别，但其生成piRNA的能力存在显著差异。Rhino是异染色质蛋白1（heterochromatic protein 1，HP1）的旁系同源蛋白，仅在生殖系中表达，且与所有端粒转基因相关联，但其富集程度与转基因piRNA的丰度呈正相关。这种异质性可能由端粒反转录转座子的序列特性所决定。与非端粒生殖系异染色质piRNA簇不同，piRNA缺失会导致端粒区域的HP1、Rhino以及三甲基化组蛋白H3赖氨酸9水平显著降低。因此，piRNA的存在是维持生殖系端粒染色质稳态的必要条件。此外，piRNA缺失会使端粒从核周向核内部移位，但不影响端粒末端加帽。对端粒相关序列（telomere-associated sequences，TASs）染色质的分析显示其具有极强的组织特异性：在生殖系中，TASs富集HP1与Rhino；而在体细胞组织中，TASs则受多梳家族蛋白的抑制调控。结论 piRNAs在生殖系端粒染色质组装以及核内端粒定位过程中发挥着至关重要的作用。端粒阵列与TASs属于一类独特的、依赖Rhino的piRNA簇，其转录本同时兼具piRNA前体与piRNA唯一靶标的双重功能。端粒染色质对piRNA缺失高度敏感，这暗示生殖系中存在一种依赖于端粒完整性的新型发育检验点。整体实验设计：对4株黑腹果蝇（Drosophila melanogaster）转基因品系卵巢中19~29 nt的小RNA组分进行测序，测序平台为Illumina HiSeq 2000。