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Transposable elements are genomic parasites that expand within and spread between genomes 1 . PIWI proteins control transposon activity, notably in the germline 2 , 3 . These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5′-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans . Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5′-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m 7 G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors 4 , and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5′-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity 5 , exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements. The endoribonuclease PUCH, a trimer of Schlafen-like-domain proteins, initiates piRNA processing in the nematode Caenorhabditis elegans through 5′-end piRNA precursor cleavage.

In eukaryotes, small RNA guides, such as small interfering RNAs and microRNAs, direct AGO-clade Argonaute proteins to regulate gene expression and defend the genome against external threats. Only animals make a second clade of Argonaute proteins: PIWI proteins. PIWI proteins use PIWI-interacting RNAs (piRNAs) to repress complementary transposon transcripts 1 , 2 . In theory, transposons could evade silencing through target site mutations that reduce piRNA complementarity. Here we report that, unlike AGO proteins, PIWI proteins efficiently cleave transcripts that are only partially paired to their piRNA guides. Examination of target binding and cleavage by mouse and sponge PIWI proteins revealed that PIWI slicing tolerates mismatches to any target nucleotide, including those flanking the scissile phosphate. Even canonical seed pairing is dispensable for PIWI binding or cleavage, unlike plant and animal AGOs, which require uninterrupted target pairing from the seed to the nucleotides past the scissile bond 3 , 4 . PIWI proteins are therefore better equipped than AGO proteins to target newly acquired or rapidly diverging endogenous transposons without recourse to new small RNA guides. Conversely, the minimum requirements for PIWI slicing are sufficient to avoid inadvertent silencing of host RNAs. Our results demonstrate the biological advantage of PIWI over AGO proteins in defending the genome against transposons and suggest an explanation for why the piRNA pathway was retained in animal evolution. Of the two types of Argonaute proteins produced by animals, AGO and PIWI, PIWI proteins can bind RNAs with less complementarity, enabling efficient silencing of transposons without the need to produce new RNA guides.

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns 1 , and also functional 2 or non-functional chimeric transcripts 3 . The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1’s conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline. SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements yet maintaining splicing integrity, via prevention of the exonization of previously integrated transposable elements, a major constituent of human genes.

Small non-coding RNAs (sRNAs) act as post-transcriptional regulators and protectors of genomic integrity. Environmental stress and genomic variation can both alter the sRNA composition in sperm, leading to altered offspring phenotypes. Commercial pig breeds, which have undergone years of selective breeding, vary in purpose: some are optimized for meat production, others for high reproductive capacity – traits that are often antagonistic. This study aims to characterize and compare the sperm sRNA composition in four commercial pig breeds, two used for meat production (sire lines) and two for reproductive purposes (dam lines). We find that the two breeds propagated for reproduction traits (Landrace, Yorkshire) show high variability in PIWI-interacting RNA (piRNA) which target transposable elements. In contrast, the two meat production breeds (Duroc, Hampshire) were enriched in mitochondrial ribosomal RNA-derived sRNA (mt rsRNA). These trends may reflect an emphasis on genome protection among reproductive breeds, and on mitochondrial function and metabolic output in meat production breeds. Other notable differences included amongst the miRNAs, which were associated with numerous developmental and metabolic processes. Our study investigates the unique sRNA profiles in boar sperm associated with different breeding objectives, highlighting possible epigenetic mechanisms that could be leveraged in selective breeding programs to optimize fertility, growth and offspring health.

PIWI proteins maintain genome integrity by piRNA-guided cleavage of complementary RNA targets. While Cleave-N’-Seq (CNS-seq) has advanced our understanding of PIWI targeting logic through quantitative mapping of cleavage rates and pairing rules, its labor-intensive workflows hinder systematic exploration of sequence determinants. Here, we present PAIRNet, a deep learning framework that predicts PIWI-mediated RNA cleavage rates by explicitly modeling guide-target interactions. Recognizing that interaction geometry, not just sequence, dictates cleavage efficiency, PAIRNet integrates biochemical insights with computational innovation: it encodes pairing states, mismatch types, insertions, and deletions alongside learnable positional embeddings to quantify spatial dependencies; employs a hybrid CNN-Transformer architecture prioritizing duplex dynamics over static sequence features to resolve both local catalytic motifs (e.g., contiguous base-pairing at g10–g11) and distal structural perturbations; and incorporates interpretability modules (saliency maps, counterfactual analysis) to link interaction patterns to biochemical insights and uncover position-specific cleavage rules. Validated across four PIWI-guide datasets, PAIRNet consistently ranks among the top two performers in all experimental conditions, achieving the most pronounced relative improvements in PCC, 34.7% for MILI and 14.6% for MIWI, over second-ranking methods. Critically, PAIRNet recapitulates key biological principles—stringent complementarity at catalytic residues (g10–g11) and tolerance for 3’ mismatches—aligning with structural studies of PIWI dynamics. By bridging biochemical precision with computational scalability, PAIRNet establishes a roadmap for designing high-specificity piRNA silencing tools while accelerating mechanistic studies of RNA-guided genome defense.

The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host–pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis . The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with Ef Piwi piRISC, consistent with their ability to capture targets more efficiently than Ef Piwi piRISC. In the presence of target, the seed gate—which enforces seed–target fidelity in microRNA RISC—adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed–target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA–target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species. This study provides structural and biochemical insight into how mammalian PIWI proteins use a limited supply of piRNAs to silence a vast array of ever-evolving transposons in the germline.

The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice 1 . piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. 2 – 5 ). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. 6 ). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1–SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation. In male mouse germline development, the precise DNA methylation of young, active transposons requires a two-step process in which SPIN1 and SPOCD1 mark young LINE1 elements before the piRNA pathway triggers DNA methylation.

Predicting the interactions between piRNA sequences and mRNA sequences is central to understanding post-transcriptional regulation in the germline and to the design of perturbations that modulate PIWI (P-element-induced wimpy testis) guided silencing. Any improvement in the accuracy of predictions about RNA sequence interactions is vital to the enhancements that can be made in many critical medical fields. This paper proposes rbpCNN, a lightweight convolutional neural network (CNN) that augments nucleotide-pair encoding with biophysically motivated interaction channels prior to learning. By adding one compatibility channel, two helix-run channels, one positional channel, and one structural channel, we aimed to support the predictions of the CNN layer and improve prediction accuracy. The resulting network is lightweight and achieves strong performance relative to existing solutions on this benchmark. Experimental results showed that rbpCNN achieves an AUC (area under the receiver operating characteristic curve) of 96.55% and an accuracy of 90.66% in fivefold validation, and an AUC of 94.19% and an accuracy of 86.74% on a separate, fully independent external dataset, with performance competitive with and, in several metrics, exceeding previously reported results on the same benchmark.

The piwi-interacting RNAs (piRNAs) are essential for controlling transposable elements (TEs) activity in germ cells, yet their expression dynamics and functions during human oogenesis remain poorly understood. Here, we simultaneously profile small and long RNAs in individual human oocytes across four developmental stages. piRNAs, especially PIWIL3-associated short piRNAs, are the predominant small non-coding RNAs during human oogenesis. A marked increase in short-piRNAs coincide with a global reduction of TE expression, particularly LINE-1 and endogenous retroviruses (ERVs). In contrast, PIWIL1- and PIWIL2-associated long piRNAs correlate with the downregulation of certain specific ERV subfamilies. Genomic analyses reveal that highly productive piRNA clusters have evolved asymmetric antisense insertion bias toward LINE-1 and ERVs, enabling TE families-specific regulation. Together, our study provides a valuable single-cell dataset of small and long RNA co-expression landscapes in developing human oocytes and reveals coordinated yet distinct roles of different PIWI/piRNA classes in TE repression during human oogenesis, with short-piRNAs acting as the primary and broad-spectrum suppressors, and long-piRNAs providing coordinated ERV-specific repression. The roles of the piRNA pathway in human oocytes are poorly understood. Here, the authors profile small and long RNAs in single oocytes and show that short piRNAs broadly repress transposable elements, while long piRNAs provide coordinated, endogenous retrovirus-specific repression.

Female infertility often arises from oogenic defects, yet the underlying molecular mechanisms remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis, but its role in mammalian female fertility remains unclear, partly due to reliance on mouse models lacking PIWIL3. PIWIL3 exits in most other placental mammals and is highly expressed in human oocytes, but its function remains largely unexplored. Here, we show that rabbit PIWIL3 closely resembles its human counterpart and is the predominant PIWI protein in oocytes. Using CRISPR-Cas9 knockout, we demonstrate that PIWIL3 is essential for female fertility in rabbits, its loss leads to severe defects in oogenesis. Embryos lacking maternal PIWIL3 arrest by the 8-cell stage. Mechanistically, PIWIL3 binds ~18-nucleotide piRNAs, supports piRNA biogenesis, and regulates transcriptomic, proteomic, and transposable element dynamics during oocyte maturation and early embryogenesis. These findings establish PIWIL3 as an essential regulator of female fertility in non-rodent mammals, potentially including humans. The PIWI–piRNA pathway is essential for gametogenesis, with PIWIL3 highly expressed in human oocytes but absent in mice. Using a rabbit model, the authors show that PIWIL3 is required for female fertility by promoting piRNA production and broadly regulating the oocyte transcriptome and proteome. Loss of PIWIL3 impairs oocyte maturation and arrests early embryonic development.