Julius Brennecke

Eukaryotic genomes generate a plethora of polyadenylated (pA ⁺ ) RNAs ^1,2 , which are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA ⁺ RNPs, including protein-coding RNPs, are exported to the cytoplasm, whereas transcripts within non-functional pA ⁺ RNPs are degraded in the nucleus ^1–4 . How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56 (also known as DDX39B) is a central component of functional pA ⁺ RNPs, and promotes their docking to the nuclear pore complex-anchored TREX-2 ^5,6 , which triggers transcript release from UAP56 to facilitate export ⁷ . Here we reveal that the poly(A) tail exosome targeting (PAXT) connection ⁸ binds a TREX-2-like module, which releases pA ⁺ RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8–PCID2–SEM1 trimer, which we show is structurally and biochemically equivalent to the central GANP–PCID2–SEM1 trimer of TREX-2. Mutagenesis and transcriptomic data demonstrate that the nuclear fate of pA ⁺ RNPs is governed by the contending actions of nucleoplasmic PAXT and nuclear pore complex-associated TREX-2, which interpret RNA-bound UAP56 as a signal for RNA decay or export, respectively. As RNA targets of PAXT are generally short and intron-poor, we propose an overall model for pA ⁺ RNP fate determination whereby the distinct sub-nuclear localizations of PAXT and TREX-2 govern the degradation of short non-functional pA ⁺ RNAs while allowing export of their longer and functional counterparts.

Background: Accurate reconstruction of repetitive genomic regions is essential to understand small RNA–mediated transposon silencing. In Drosophila ovarian somatic cells (OSCs), a primary model for nuclear PIWI–piRNA biology, reliance on the D. melanogaster reference (dm6) obscures cell-line–specific transposon insertions and the architecture of piRNA source loci. Results: We generated a highly contiguous de novo assembly of the OSC genome that reveals a transposon landscape diverging substantially from dm6. The assembly fully resolves the ∼700 kb flamenco locus, the principal piRNA cluster in OSCs, and yields a genome-wide map of transposon insertions. By integrating this assembly with functional genomics datasets, we define OSC piRNA source loci, show that cluster sequence composition determines transposon piRNA output, and demonstrate the widespread effects of Piwi on heterochromatin formation. Conclusions: The OSC assembly establishes a precise genomic framework for interpreting piRNA and chromatin data in this model system. To enable community use of this resource for studies of transposon control and piRNA biology, we provide an integrated genome browser and data-sharing platform.

How host organisms adapt their defense systems to newly invading transposable elements remains poorly understood. Here, we show how Drosophila melanogaster acquired PIWI-interacting RNA (piRNA)-mediated immunity against the endogenous retrovirus Tirant . We uncover two distinct modes of de novo piRNA biogenesis by combining genetics, small RNA profiling, and population genomics. The primary route involves antisense insertions into the flamenco cluster, a master locus for transposon control. Unexpectedly, a second, equally potent mechanism arises from antisense Tirant insertions within host gene 3′ UTRs. This process requires host gene transcription but is independent of host gene identity. Our findings challenge prevailing models that tie piRNA precursor specification to genomic origin or nuclear RNA processing context. Instead, they reveal a flexible mechanism that turns a critical vulnerability of transposons into an advantage for the host. When transposition occurs into host gene exons, chimeric antisense transcripts are exported to the cytoplasm, inadvertently initiating piRNA production and enabling rapid, adaptive genome defense against new invaders.

How piRNA-mediated genome defense achieves specificity against transposons while sampling a complex transcriptome has remained unresolved. Here we show that piRNA biogenesis operates through pervasive, non-specific sampling of cytoplasmic RNAs, with specificity imposed by tissue-specific molecular modules that exploit intrinsic vulnerabilities of transposons. In Drosophila somatic cells, the specificity factor Yb steers basal processing towards uridine-rich RNAs—automatically capturing antisense retrotransposon transcripts due to their intrinsically adenosine-biased genomes. In germline cells lacking Yb, basal sampling generates naïve piRNAs loaded into catalytically active Argonaute proteins, which trigger autocatalytic ping-pong amplification upon encountering complementary targets. In both contexts, transposon mobility facilitates the production of antisense RNAs that enable either biased processing or amplification. Thus, piRNA clusters, long associated with pathway specificity, act as sources of transposon antisense sequences, while specificity arises from layering distinct molecular mechanisms onto a shared foundation of indiscriminate transcript sampling, enabling robust and adaptable genome defense without predefined templates.

Nuclear Argonaute proteins safeguard genome integrity by directing transcriptional silencing and heterochromatin formation at transposon loci. Yet it remains unclear how Argonautes enforce robust repression while relying on target transcription for their own recruitment. Here we show that transposon silencing by the Drosophila nuclear Piwi-piRNA pathway requires degradation of target RNA by the nuclear exosome. Using proximity proteomics at endogenous Piwi target sites, we identify two previously uncharacterized paralogs, TEsup-1 and TEsup-2, as essential cofactors for Piwi-mediated silencing. TEsup proteins act in part by engaging nuclear exosome adaptor complexes at piRNA-targeted transcripts through a domain that recognizes proline-rich peptides. Disruption of the Piwi-TEsup-exosome axis leads to accumulation and nuclear export of piRNA-targeted transposon RNAs. Notably, the P-element, which evades heterochromatin-based repression, is silenced primarily through this RNA-decay pathway. Thus, the nuclear piRNA pathway couples target recognition to RNA degradation, reconciling small RNA-guided heterochromatin formation with ongoing transcription at target loci.

The nuclear export of mRNA is an important step in eukaryotic gene expression ¹ . Despite recent molecular insights into how newly transcribed mRNAs are packaged into ribonucleoprotein complexes (mRNPs) ^2,3 , the subsequent events that govern mRNA export are poorly understood. Here we uncover the molecular basis underlying key events of human mRNA export, including the remodelling of mRNP-bound transcription–export complexes (TREX), the formation of export-competent mRNPs, the docking of mRNPs at the nuclear pore complex (NPC), and the release of mRNPs at the NPC to initiate their export. Our biochemical and structural data show that the ATPase UAP56 (also known as DDX39) acts as a central molecular switch that directs nucleoplasmic mRNPs from TREX to NPC-anchored TREX-2 complexes through its ATP-gated mRNA-binding cycle. Collectively, these findings establish a mechanistic framework for a general and evolutionarily conserved mRNA export pathway.

Accurate genome assemblies are critical for understanding small RNA-mediated genome defense. In animals, the PIWI-interacting RNA (piRNA) pathway protects genome integrity by silencing transposable elements. Studying how piRNAs are generated and how they guide heterochromatin formation requires complete reconstruction of genomic piRNA source loci and detailed transposon maps. Here, we present a high-quality de novo genome assembly of Drosophila melanogaster ovarian somatic cells (OSCs), a widely used cell line that recapitulates nuclear piRNA biology. The OSC genome differs substantially from the reference genome, with major differences in transposon content and piRNA cluster composition. Our assembly resolves the 700 kb flamenco locus, the primary piRNA cluster in OSCs, and provides a genome-wide transposon map. Using this resource, we characterize piRNA source loci, reveal how piRNA cluster composition determines transposon-derived piRNA profiles, and clarify the widespread impact of the nuclear piRNA pathway on heterochromatin. Finally, we provide an open platform for integrating user-generated datasets with the OSC genome, creating a community resource for studying transposon control and piRNA biology.