RNA Decay via the Nuclear Exosome is Essential for Piwi-Mediated Transposon Silencing [TT-seq]

Small RNA pathways in the nucleus safeguard genome integrity by directing transcriptional silencing and heterochromatin formation at transposon loci. Yet, how these pathways achieve effective repression despite requiring transcription for Argonaute targeting has remained unclear. Here, we show that in Drosophila, the Piwi–piRNA pathway enforces transposon silencing through RNA degradation by the nuclear RNA exosome. Using comprehensive proximity proteomics at piRNA target sites combined with genetic analyses, we identify two previously uncharacterized paralogs, TEsup-1 and TEsup-2, which connect Piwi to nuclear exosome adaptor complexes via a distinct binding module for proline-rich peptides. Disruption of this Piwi–TEsup–exosome axis leads to accumulation and nuclear export of transposon RNAs. Strikingly, transposons that evade heterochromatin-based repression, exemplified by the P-element, are repressed primarily through this RNA-decay pathway. These findings reveal a molecular mechanism by which the nuclear piRNA-pathway couples target recognition to RNA degradation, providing a framework that reconciles small RNA-guided heterochromatin formation with ongoing transcription at target loci. Overall design: Transient transcriptome sequencing (TT-seq) analyis of nascent transrption changes in cultured ovarian somatic cells (OSCs) after siRNA mediated depletion of dZfc3h1, dZcchc7, dZcchc8, or dZfc3h1&dZcchc8, compring to control knockdown.细胞核内的小RNA通路通过介导转座子位点的转录沉默与异染色质形成，维系基因组完整性。然而，尽管阿戈奈特（Argonaute）蛋白的靶向过程依赖转录，此类通路如何实现高效的转座子沉默仍悬而未决。 本研究以果蝇（Drosophila）为模型，发现Piwi-piRNA（Piwi相互作用RNA）通路可通过核RNA外泌体介导的RNA降解，实现转座子的有效沉默。研究人员结合piRNA靶标位点的全范围邻近蛋白质组学分析与遗传分析，鉴定出两个此前未被表征的旁系同源基因TEsup-1与TEsup-2：二者通过识别富脯氨酸肽的独特结合模块，将Piwi与核外泌体衔接复合体相连。 Piwi-TEsup-外泌体通路轴的破坏会导致转座子RNA的积累与核输出。值得注意的是，以P因子（P-element）为代表的、逃避异染色质介导沉默的转座子，主要通过该RNA降解通路实现抑制。本研究揭示了细胞核piRNA通路将靶标识别与RNA降解相偶联的分子机制，为协调小RNA介导的异染色质形成与靶标位点的持续转录提供了理论框架。 实验设计：本研究通过siRNA介导分别敲低dZfc3h1、dZcchc7、dZcchc8以及dZfc3h1与dZcchc8联合敲低，对培养的卵巢体细胞（OSCs）中的新生转录变化进行瞬时转录组测序（TT-seq）分析，并以阴性敲低组作为对照。