Biology subjects

Wen, J.

Publications and source records attributed to Wen, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer

DNA methylation (5-methylcytosine, 5mC) is a major repressive epigenetic mark that can be iteratively oxidized by ten-eleven-translocation (TET) enzymes to 5-hydroxymethylcytosine (5hmC); beyond DNA, modifications of RNA can also affect transcript stability and processing, yet RNA hydroxymethylation remains unexplored in breast cancer (BC). Here, we identify a quantifiable cytoplasmic 5hmC signal in BC cell lines and, more prominently, in human BC tissue, using immunoassays and digital pathology, and confirm with an adapted, RNase-sensitive, RNA-specific ELISA that this signal reflects hydroxymethylation of total RNA and mRNA. Cytoplasmic 5hmC was significantly enriched in triple-negative (ER-/PR-/HER2-) and grade 3 tumors, and correlated with increased cytoplasmic TET2/TET3 in tissue and TET3 upregulation in TCGA. As a leading candidate mechanism, TET3 expression was associated with RNA-processing/spliceosomal transcripts, and TET3 knockdown in three TNBC cell lines reduced a subset of these targets, independently supported across 70 DepMap breast cancer lines.

cancer biology↗

bioRxiv2026

Systematic Benchmarking of AI-Based Molecular Generation Models for Structure-Based Drug Design

Generative artificial intelligence is accelerating molecular design, yet the relative suitability of available models for different targets and stages of preclinical drug discovery remains unclear. Here we benchmarked 12 molecular generation and optimization methods across 176 curated protein-ligand systems spanning diverse therapeutic target classes, with experimentally validated ligands providing reference chemical space. The evaluated methods encompassed pocket-conditioned 3D generation, diffusion and flow-based modeling, autoregressive construction, reference-conditioned optimization and synthesis-aware design. Performance was assessed using operational robustness, chemical validity, uniqueness, molecular and scaffold diversity, quantitative estimate of drug-likeness, synthetic accessibility, docking, physicochemical and ADMET properties, and computational resource requirements. The results revealed architecture-dependent trade off such as receptor-conditioned methods exploited binding-pocket geometry, flow-based approaches enabled efficient sampling, reference-conditioned methods favored analogue generation, and synthesis-aware approaches improved chemical feasibility, but no method consistently optimized all criteria. To address the functional potential of generated molecules, we further developed a state-aware functional classifier (SAFC) that integrates molecular dynamics derived receptor ensembles, ensemble docking and protein ligand interaction graphs. SAFC provided dynamics-aware functional activity rankings for generated molecules that were partly complementary to docking, drug-likeness and synthetic accessibility scores. These findings support hybrid, stage specific deployment of generative models rather than reliance on any single architecture or evaluation metric. This study provides practical guidelines for generative AI based preclinical drug development processes.

bioinformatics↗

bioRxiv2026

Histone lysine demethylase inhibition is a disease-modifying therapy for hypertrophic cardiomyopathy

RationaleHypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by cardiac hypertrophy, fibrosis, arrhythmias, and sudden cardiac death (SCD). Although current therapies primarily target sarcomere dysfunction, the contribution of epigenetic dysregulation to HCM pathogenesis and its therapeutic potential remain poorly understood. ObjectiveTo determine whether pharmacological inhibition of histone lysine demethylases (KDMs) with JIB-04 can prevent or reverse HCM progression and to identify the underlying epigenetic mechanisms. Methods and ResultsWe evaluated the pan-KDM inhibitor JIB-04 in Myh6R403Q/+ mice carrying the murine equivalent of the pathogenic human MYH7 R403Q mutation. JIB-04 prevented disease progression, reduced cardiac hypertrophy and fibrosis, preserved cardiac function, and completely prevented SCD in cyclosporin A- accelerated HCM. JIB-04 also reversed established disease, produced sustained therapeutic benefits after drug withdrawal, and improved cardiac function in aged mice with spontaneous HCM. Bulk RNA sequencing and ATAC-seq demonstrated partial restoration of disease-associated transcriptional programs and chromatin accessibility. Proteomic analyses identified PHF2 (KDM7C) as a candidate target of JIB-04 in both mouse and human HCM hearts. PHF2 knockdown suppressed hypertrophic, inflammatory, and fibrotic gene expression in cardiomyocytes, macrophages, and fibroblasts, respectively. Human HCM hearts exhibited increased expression of multiple JIB-04-sensitive KDMs, including PHF2. In MYH7 R403Q induced pluripotent stem cell- derived cardiomyocytes, JIB-04 normalized disease-associated gene expression, restored connexin-43 membrane localization, and improved mitochondrial respiration. Although prolonged treatment induced reversible hepatomegaly with hepatic lipid accumulation, co-administration of the antioxidant N-acetylcysteine mitigated liver toxicity while preserving the therapeutic efficacy of JIB-04. ConclusionsPharmacological KDM inhibition prevents and reverses HCM through epigenetic remodeling of disease-associated transcriptional and chromatin programs. These findings identify KDM inhibition as a promising therapeutic strategy for HCM, establish PHF2 as a candidate mediator of disease pathogenesis, and support further development of KDM-targeted therapies.

physiology↗

bioRxiv2026

Presynaptic mechanism of epileptiform activities in forward-programmed human excitatory neuronal networks

Epilepsy is one of the most common neurological disorders, yet the mechanisms controlling seizure termination remain poorly understood. In particular, why rhythmic spike-wave discharges decelerate before stopping is unexplained. Here, using human iPSC-derived excitatory neurons differentiated via targeted forward-programming, we report a similar deceleration phenomenon in cultured neuronal networks. These networks exhibit glutamate-dependent, epileptiform 'super-bursts' with a slowing rhythm from ~4 Hz to ~2 Hz. Combining in silico simulations and in vitro experiments, we correlate this activity pattern with the hierarchical organization of presynaptic vesicle pools. Nested bursts link to the recycling pool (RP), and sub-bursts associate with the readily releasable pool (RRP). Decelerating RP-to-RRP vesicle translocation shortened the super-bursts, indicating that epileptiform dynamics depend heavily on this translocation process. These findings depict human neuronal networks derived from forward-programmed cells as a model for epileptology, revealing a presynaptic framework for rhythmic discharges in excitatory networks.

neuroscience↗

bioRxiv2026

Untargeted metabolomics reveals host responses and metabolites linked to host compatibility in Rafflesiaceae parasitism

Rafflesiaceae, including Rafflesia, Sapria, and Rhizanthes, are some of the rarest flowering plants in the world, known for producing the largest blooms on Earth. These holoparasites depend exclusively on Tetrastigma (Vitaceae) vines, yet the chemical basis for this host specificity is poorly understood, complicating conservation efforts. Untargeted negative-ion LC- MS metabolomics was used to profile Rafflesiaceae buds and seeds, infected Tetrastigma hosts associated with Rafflesia lagascae, R. speciosa, and Sapria himalayana, uninfected Tetrastigma hosts, and corresponding non-host species from the Philippines and Thailand. Python scripts used for data processing and visualization were developed with generative AI assistance and validated by the authors. Principal component analysis revealed distinct metabolomic profiles across samples, with significant shifts in host metabolites upon infection. Infected Tetrastigma tissues showed nominal enrichment in phenylpropanoid and gibberellin-related metabolites, suggestive of defense and hormonal pathway activation during Rafflesia infection, as well as citric acid, suggesting increased energy demand to support the parasite. Non-host Tetrastigma species showed nominal enrichment in metabolites putatively annotated as stilbenoids, resveratrol diglucosides, and condensed tannins such as procyanidin B2, compounds broadly associated with plant defense. Rafflesia seeds harbored fatty acids/oxylipins, while Rafflesia buds accumulated gallic acid derivatives, compounds also reported in galls. These findings support a chemically mediated model of host compatibility and parasitism, with implications for the ex-situ conservation of these endangered plants.

plant biology↗

bioRxiv2026

Mechanism of fatty acid uptake and inhibition in human FATP2

Fatty Acid Transport Protein 2 (FATP2) couples fatty acid uptake to intracellular activation and is associated with pathological lipid accumulation in cancer and nonalcoholic fatty liver disease. Here, we present cryo-electron microscopy structures of human FATP2 across its reaction cycle. Our structures suggest that FATP2 recruits fatty acids directly from the membrane interface through a hydrophobic tunnel. Catalysis involves a [~]130{degrees} rotation of the C-terminal domain, a transition trapped by the antihypertensive drugs isradipine and benidipine. Both drugs lock the enzyme in a thioester-forming state, but benidipine exhibits superior efficacy by extending a bulky moiety into the primary catalytic tunnel to sterically block substrate entry. Furthermore, we identify a product inhibition mechanism where excess acyl-CoA traps the enzyme, potentially limiting metabolic overload. These findings provide a structural framework for understanding vectorial fatty acid channeling and a scaffold for developing modulators of metabolic flux. HighlightsO_LICryo-EM structures of human FATP2 reveal a membrane-anchored lollipop topology C_LIO_LIEndogenous fatty acids within a hydrophobic tunnel delineate the fatty acid uptake pathway C_LIO_LIIsradipine and benidipine displace fatty acids to trap a non-productive conformation C_LIO_LIAcyl-CoA product inhibition may provide negative feedback via steric occlusion C_LI

biophysics↗

bioRxiv2026

Chromosome-level genome assembly of Calotes wangi with dynamic colour variation

The Wangs garden lizard, Calotes wangi, is a widely distributed agamid species in Southern China and Northern Vietnam and exhibits pronounced colour variation and rapid body colour change. Despite increasing interest in the genomic basis of colour variation, chromosome-level genomic resources remain limited in agamid lizards. Here, we generated a chromosome-level reference genome of C. wangi using PacBio HiFi sequencing and Hi-C scaffolding. The final genome assembly was approximately 1.66 Gb in size and comprised 6 macrochromosomes and 11 microchromosomes, with a contig N50 of 110.09 Mb and 98.9% complete BUSCO genes. A total of 20,442 protein-coding genes were annotated. Comparative genomic analyses identified 297 significantly expanded gene families, with enriched functions associated with steroid metabolism, chromatin regulation, and epigenetic processes. This high-quality genome assembly provides an important genomic resource for future studies of colour variation, phenotypic plasticity, and evolutionary diversification in agamid lizards.

evolutionary biology↗

bioRxiv2026

Redundant γc cytokines license IL-1-driven neutrophil inflammation through MEK/ERK convergence

Interleukin-1 (IL-1) is a central driver of autoinflammatory disease, yet IL-1 blockade often provides incomplete benefit in complex, neutrophil-driven conditions. Here we identify a licensing circuit in which common {gamma}-chain ({gamma}c) cytokines provide a redundant signal required for maximal IL-1-driven neutrophil inflammation. IL-1 and {gamma}c cytokines synergize to drive inflammatory cytokine production exceeding either stimulus alone, and these signals engage the MEK/ERK pathway, an effect substantially suppressed by pharmacological MEK inhibition. We validated this circuit in vivo in a mouse model of IL-1-driven neutrophil-dominant autoinflammation. Ablation of the shared {gamma}c receptor markedly prolonged survival and attenuated pathology, whereas deletion of individual {gamma}c cytokine pathways had no major effect--demonstrating in vivo necessity and functional redundancy. Analysis of public phospho-proteomic and transcriptomic datasets confirms MEK/ERK as a conserved neutrophil response to diverse inflammatory stimuli and coordinated IL-1, {gamma}c, and MEK/ERK activation in neutrophils from patients with systemic juvenile idiopathic arthritis (sJIA) and in lesional skin from hidradenitis suppurativa. Together, these findings define a signaling architecture in which redundant {gamma}c inputs enhance MEK/ERK-dependent inflammatory output, identify the {gamma}c receptor as an in vivo disease-modifying node, and position MEK/ERK as a mechanistically grounded therapeutic target. eTOC SummaryLorenzo et al. show that common {gamma}-chain ({gamma}c) cytokines provide redundant licensing signals that amplify IL-1-driven neutrophil inflammation through MEK/ERK convergence. Blocking any single {gamma}c cytokine fails to suppress disease, but ablating the shared {gamma}c receptor or inhibiting MEK/ERK markedly attenuates pathology, identifying these nodes as therapeutic targets in autoinflammatory disease.

immunology↗

bioRxiv2026

FAM134B isoform 2/RETREG1-2 defines a calnexin-TOLLIP-coupled ER-phagy pathway that restricts Ebola virus glycoprotein and is antagonized by VP40 through macro-autophagy

Selective autophagy of the endoplasmic reticulum (ER-phagy) is critical for ER proteostasis and host defense, yet how ER quality-control pathways interface with ER-phagy to restrict viral glycoproteins remains poorly defined. Previously, the 1st known ER-phagy receptor gene RETREG1 (RETR1)/FAM134B gene was reported to restrict Ebola virus (EBOV) replication in vivo by inhibiting the viral glycoprotein (GP) and viral protein 40 kDa (VP40) expression, but this mechanism remains unknown. Here, we identify the truncated RETR1/FAM134B isoform 2 (RETR1-2), but not its full-length protein RETR1, as an ER-phagy receptor that targets EBOV-GP for degradation. RETR1-2 broadly triggers GP degradation across ebolavirus species but not Marburg virus and inhibits EBOV replication. Mechanistically, RETR1-2 recognizes EBOV-GP via its luminal domain, undergoes GP-induced oligomerization, and directs GP-containing ER membranes to lysosomes through canonical macro-autophagy. Using unbiased mass spectrometry, we identified TOLLIP as the key cytoplasm adaptor for RETR1-2, which also requires cooperation with the ER chaperone calnexin for EBOV-GP degradation. Notably, the PI3P-binding C2 domain of TOLLIP mediates its interaction with RETR1-2, and the EBOV-GP degradation occurs independently of ubiquitination, revealing an unexpected role for TOLLIP in ER-phagy. Furthermore, EBOV-VP40 antagonizes this pathway by selectively targeting RETR1-2 for macroautophagic degradation independently of TOLLIP, thereby restoring GP expression and viral infectivity. Nevertheless, RETR1-2 reciprocally degrades VP40 via a similar mechanism. Together, these findings define a calnexin-TOLLIP-RETR1-2 axis that links ER quality control to ER-phagy-mediated antiviral restriction and uncover a reciprocal host-virus arms race centered on selective macro-autophagy.

microbiology↗

bioRxiv2026

Medicago truncatula Iron-chaperone 1 (ICHAP1) is required for symbiotic nitrogen fixation

Hundreds of proteins in the cell require iron (Fe) or Fe-containing cofactors to function. However, how Fe2+ or Fe3+ are specifically allocated to each of these proteins in plant cells remains largely unknown. It has been proposed that Fe metalation could be driven by specific interactions with Fe-shuttling proteins known as Fe-chaperones. Here, we present the first family of plant Fe2+-chaperones (ICHAPs) with orthologues in dicots and monocots. The role of these proteins in Fe distribution to Fe-dependent metabolic processes has been illustrated using symbiotic nitrogen fixation in Medicago truncatula root nodules. ICHAP1 is a soluble Fe2+-binding protein that interacts with plasma membrane Fe2+ transporter NRAMP1, but not with symbiosome Fe2+-transporters. ICHAP1 mutants present altered Fe distribution in cells and they cannot fix nitrogen. A second family member, ICHAP2 is required to target Fe2+ to symbiosomes, as it accepts Fe2+ from ICHAP1 and interacts with symbiosome Fe2+-importer VTL8, but not with NRAMP1. These results indicate a path for Fe2+ allocation from the plasma membrane to the symbiosome through specific protein-protein interactions and Fe2+ exchange from NRAMP1 to ICHAP1, to ICHAP2, and to VTL8.

plant biology↗

bioRxiv2026

RNAElectra: An ELECTRA-Style RNA Foundation Model for RNA Regulatory Inference

BackgroundRNA regulation programs gene expression through sequence-encoded mechanisms--including RNA structure formation, protein binding, chemical modification, and RNA-RNA targeting--whose rules span nucleotide-scale motifs and longer-range context. RNA foundation models aim to learn transferable representations from large RNA corpora, but most rely on masked language modeling (MLM), where the model loss is computed on a small subset of positions and training relies on artificially corrupted inputs that are absent at downstream inference, introducing a pretraining-downstream discrepancy. ResultsWe introduce RNAElectra, a single-nucleotide-resolution RNA foundation model pretrained on diverse non-coding RNAs from RNAcentral using ELECTRA-style replaced-token detection (RTD). RTD trains a discriminator with a loss defined over all input positions on realistically corrupted sequences, providing dense supervision that better aligns pretraining with sequence-to-function fine-tuning. RNAElectra combines nucleotide-resolution tokenization with an efficient attention design to capture local regulatory motifs and longer-range dependencies within a single reusable backbone. Using a unified, sequence-only fine-tuning pipeline without task-specific architectures or auxiliary inputs, RNAElectra demonstrates strong cross-task generalization across benchmarks and downstream evaluations spanning RNA structure and function, RNA-protein and RNA-RNA interactions, RNA modification landscapes, and quantitative regulatory readouts such as translation efficiency and mRNA stability, outperforming widely used RNA foundation model baselines on the majority of evaluated tasks. In addition to predictive performance, RNAElectra supports interpretability by enabling analysis of learned representations and sequence determinants underlying model predictions. ConclusionRNAElectra establishes RTD pretraining as a practical alternative to MLM for RNA foundation modeling, coupling dense position-wise supervision with an efficient architecture to deliver broadly transferable RNA representations. This framework provides a reusable backbone for RNA regulatory prediction and supports sequence-level RNA engineering and design.

bioinformatics↗

bioRxiv2026

Transformation and recombination of neural information in a brain network

Mammalian brain function relies on integrated interactions among interconnected neural structures. The principles by which regional activity is transformed into projection-specific signals and then recombined at targets elsewhere in the brain are fundamental to brain processing, but remain poorly understood. Here we study these phenomena using a genetically encoded probe that provides neurophysiological readouts from virally labeled projections on a brain-wide scale via functional magnetic resonance imaging (fMRI). By analyzing outputs from thalamic and cortical somatosensory processing regions in rats, we find that projection-specific neural population activity undergoes shifts in tuning and temporal characteristics as it emanates from source regions. Patterns of neural information flow to targeted brain structures reconfigure under different conditions of stimulation and rest, contrasting with intrinsic fMRI functional connectivity profiles, which remain constant. Excitatory and inhibitory projections are coactivated during stimulation, but their relative response amplitudes change dynamically between stimulus conditions and across repeated stimuli, suggesting mechanistic roles for network-wide shifts in excitation/inhibition balance. Our results thus reveal how information flow throughout a neural system reshapes to promote stimulus selectivity and provide underpinnings of large-scale brain phenomena.

neuroscience↗

bioRxiv2026

A new mRNA antigen vaccine induces potent B and T cell responses and in vivo protection against SARS-CoV-2

The SARS-CoV-2 mRNA vaccine provides effective protection against viral infection and severe disease by inducing efficient adaptive immunity. However, vaccine efficacy is decreased against emerging variants, and immune memory is relatively short-lived. Here, we added new T cell epitopes to the RBD (receptor-binding domain) mRNA vaccine and identified a SARS-CoV-2 membrane epitope that significantly improved vaccine-induced immunity and protection in vivo. That new vaccine, designated G1-C, induced 8.2-fold higher levels of RBD-specific antibodies than did RBD and enhanced spike-specific T cell and B cell responses. Remarkably, the G1-C modulated hematopoietic stem cell (HSC) differentiation and increased levels of B and NK cells by regulating multiple signaling pathways in bone marrow potentially via Fos, Klf4, and Klf6 transcription factors. Altogether, these findings identify a new vaccine candidate to control viral infection by affecting the lymphoid-myeloid lineage bias and suggest the potential role of T cell epitopes in vaccine design and development.

immunology↗

bioRxiv2026

Macrophage SLC9A1 Links Endocytic Trafficking to Innate Immune Activation in Myocardial Injury

Excessive innate immune activation drives adverse remodeling after myocardial infarction (MI), yet the upstream mechanisms by which macrophages sense ischemic danger signals remain poorly defined. Here we tested whether macropinocytosis functions as a mediator of post-ischemic inflammation and whether the Na/H exchanger SLC9A1 links membrane ion transport to innate immune activation in the injured heart. Macropinocytosis was robustly activated in infarct-associated macrophages, which are the predominant cell type with the macropinocytotic activity in the injured heart. Pharmacologic inhibition of macropinocytosis with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) improved cardiac function and attenuated post-MI remodeling. EIPA also attenuated cardiac inflammatory responses induced by systemic lipopolysaccharide and Poly(I:C). To define macrophage-intrinsic mechanisms, we generated monocyte- and monocyte-derived macrophage-specific Slc9a1 knockout mice. Genetic deletion of Slc9a1 recapitulated the cardioprotective effects of EIPA and markedly suppressed interferon-stimulated gene programs in infarct-associated macrophages, as revealed by single-cell RNA sequencing. Mechanistically, SLC9A1 promoted endocytic uptake of Poly(I:C) acid and enhanced endosome-dependent inflammatory signaling. Together, these findings identify macrophage macropinocytosis as a regulator of innate immune activation after MI and reveal SLC9A1 as a previously unrecognized link between membrane ion transport and inflammatory signaling in the injured heart. Targeting SLC9A1-dependent membrane trafficking pathways may therefore represent a strategy to limit maladaptive inflammation in ischemic heart disease.

immunology↗

bioRxiv2026

Identification and Characterization of Metastasis-initiating cells

Metastasis, the primary cause of cancer-related mortality, is a dynamic and complex process driven by a subset of cells known as metastasis-initiating cells (MICs). Accurate identification of MICs is therefore critical for metastasis diagnosis and therapeutic decision-making. However, current approaches rely either on mouse tracing experiments, which are difficult to translate to human systems, or on indirect strategies such as stemness, trajectory, pathway, and biomarker analyses that often yield inconsistent results. To address these limitations, we propose scMIC, a computational framework designed to explicitly and reliably identify MICs from single-cell data (available at https://github.com/swu13/scMIC). scMIC integrates an embedding-based representation, unbalanced optimal transport, and a top-k selection strategy to robustly capture metastasis-initiating potential. The framework was validated and applied across multiple cancer types, species, and multi-omics datasets. Our results demonstrate the reliability of scMIC for MIC identification, its potential clinical utility in metastasis prognosis, and its effectiveness in discovering metastasis-related gene programs and molecular biomarkers. Elucidating the mechanisms of metastasis initiation not only advances our understanding of metastatic progression but also enables the development of therapeutic strategies that target the more aggressive MIC population rather than non-MICs, thereby avoiding unintended increases in metastatic risk. Collectively, scMIC provides a powerful tool for cancer metastasis research and drug discovery.

bioinformatics↗

bioRxiv2026

Tailored Cell Cycle Modulation Enhances AAV Manufacturing: Balancing Arrest with Adaptive Stress Responses

Recombinant adeno-associated virus (rAAV) vectors show therapeutic potential, but their biomanufacturing is limited by low yields and high costs. Host cell-cycle modulation is emerging as a promising strategy to enhance rAAV production. Two G2/M phase-arresting small molecules, ABT-751, a microtubule inhibitor, and helenalin, a thiol-reactive sesquiterpene lactone, were applied post-transfection in HEK293 cells to evaluate how cell-cycle arrest and stress pathways influence rAAV yields. ABT-751 induced G2/M arrest with minimal cytotoxicity, leading to a near five-fold increase in rAAV vector genomes across multiple serotypes and production platforms. Helenalin caused G2/M arrest, yet suppressed rAAV production. Comparative transcriptomic profiling (RNA-Seq) revealed that helenalin altered expression of a widespread set of genes (4,579) compared to control, characterized by rampant p53, ferroptosis, and endoplasmic reticulum dysregulation that overflowed into unfolded protein response with CHOP induction and apoptosis. ABT-751 elicited a more moderate, targeted response (1,895 differentially expressed genes) in a similar subset of pathways, including compensatory mechanisms mitigating oxidative stress. Together, these findings indicate that cell-cycle arrest alone is insufficient to improve rAAV yield. Indeed, tailored cell-cycle modulation, coupled with balanced activation of cellular stress pathways, can enhance rAAV manufacturing efficiency, facilitating more scalable and cost-effective gene therapy production strategies for the future.

bioengineering↗

bioRxiv2025

Recurrent expansion and rapid evolution of the Drosophilid RNAi pathway in testis

Multiple classes of selfish genetic elements, including transposable elements, meiotic drivers, and viruses, are suppressed by small interfering RNAs (siRNAs) that guide RNA interference (RNAi). These are interlocked within continually evolving genetic conflicts that are characterized by extremely rapid dynamics of change in both sequence and copy number. Indeed, it was shown that across Drosophila, the core RNAi machinery evolves under positive selection, and that the RNAi effector AGO2 has additional copies in some species. This contrasts with the core microRNA (miRNA) machinery in Drosophila, which evolves under negative selection and maintains one-to-one orthologs not only amongst flies, but even to mammals. Here, we analyze >300 long read genome assemblies of Drosophila species to generalize these attributes. Not only do we find recurrent expansion of AGO2 across [~]100 species, including lineages with ongoing amplification of AGO2, we also find dozens of species with extra copies of the other core RNAi factors, r2d2 and dicer-2. In many cases, these additional RNAi factor copies co-exist, or are nested, within a lineage bearing an ancestral expansion of AGO2. Transcriptome data provide evidence that core RNAi factors, including certain lineage-specific copies, are biased for testis expression. Finally, we use small RNA data to annotate hairpin RNAs (hpRNAs) in D. pseudoobscura, one of the species with prominent amplification of RNAi factors. We find rampant de novo hpRNA loci in this species, whose siRNAs are predominantly expressed in testis. All together, these findings highlight evolutionary plasticity of the fly RNAi pathway and affirm that it is preferentially deployed in the germline of the heterogametic sex. When considered alongside abundant genetic data for recurrent sex ratio meiotic drive against the Y chromosome, these genomic data strongly imply that a fundamental role of endogenous RNAi is to control sex chromosome conflicts.

evolutionary biology↗

bioRxiv2025

The 9p21.3 Coronary Artery Disease Risk Locus Modulates Vascular Cell-State Transitions via Enhancer-Driven Regulation of MTAP

The 9p21.3 locus is the strongest genetic association with coronary artery disease (CAD), yet its causal mechanisms remain unresolved. We map the regulatory architecture of 9p21.3 in disease-relevant vascular cells, identifying 12 enhancers within the CAD risk haplotype that respond dynamically to inflammatory and metabolic stress in fibroblasts and smooth muscle cells. These activated states are enriched for CAD heritability, implicating stress-responsive vascular wall cells in disease pathophysiology. Dense CRISPRi tiling integrated with fine-mapping and genomic constraint across >500,000 individuals nominates MTAP as the effector gene, with rs1537371 as a likely causal variant. Perturbation and multi-modal analyses show that MTAP loss induces pro-fibrotic and angiogenic programs and sensitizes vascular cells to TGF-{beta}-driven pathological transitions. Our findings reveal a vascular-specific enhancer network through which noncoding variation at 9p21.3 modulates CAD risk via MTAP--a previously unrecognized regulator of vascular remodeling located 269 kb from the risk haplotype. HighlightsO_LI9p21.3 CAD risk locus harbors 12 functional enhancers active in vascular wall cell types C_LIO_LICAD heritability is enriched in stressed vascular fibroblasts and smooth muscle cells C_LIO_LIHigh-resolution CRISPRi-MAC-seq maps 9p21.3 enhancer-gene and SNP-gene interactions C_LIO_LIMTAP identified as a causal effector gene regulated by 9p21.3 fine-mapped and constrained variants C_LIO_LIMTAP modulates TGF-{beta}-driven pathological vascular cell transitions C_LI

genomics↗