Biology subjects

Xu, H.

Publications and source records attributed to Xu, H..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Structural mechanism of lipid modulation of pentameric ligand-gated ion channel activity

Pentameric ligand-gated ion channels (pLGICs) are sensitive to the lipid environment. However, the structural mechanism of how specific lipids support the agonist response of any pLGIC is poorly understood. Using the model pLGIC, ELIC (Erwinia ligand-gated ion channel), we find that phosphatidylethanolamine (PE) or cardiolipin (CL) are sufficient to support activation of a non-desensitizing mutant called ELIC5. Cryo-EM structures of unliganded and agonist-bound ELIC5 in the absence of PE or CL show increased structural heterogeneity and destabilization of the resting and open-channel states. Importantly, the unliganded structure of ELIC5 in a phosphatidylcholine (PC)-only environment shows variability that resembles agonist-induced changes. The structures also reveal a CL binding site at an outer leaflet M3-M4 site. Together with functional measurements in asymmetric liposomes and coarse-grained molecular dynamics simulations, the data indicate that CL supports ELIC activity by binding to this M3-M4 site thereby stabilizing an agonist-responsive resting state of the channel.

biophysics↗

bioRxiv2025

Fluid-Niche and Microglial Signatures Prime Robust Intraventricular Macrophage Response to Blood During Brain Development

Intraventricular macrophages (IVMs) reside in cerebrospinal fluid (CSF) and are considered a border-associated macrophage (BAM) population in the brain. Although they represent the first line of defense against intraventricular challenges, their developmental roles and responses to injury are poorly understood. This knowledge is relevant for conditions including neonatal intraventricular hemorrhage (IVH), where blood extravasates into brain ventricles, leading to life-long negative sequelae including cerebral palsy and hydrocephalus. Here, we show that IVMs are first responders to blood in developing brain ventricles, phagocytosing red blood cells and upregulating iron-processing machinery. Live imaging of developing mouse ventricles and choroid plexus revealed that IVMs are dynamic and morphologically distinct from non-IVM macrophages. Their transcriptional profiles distinguish them from other BAMs as they also exhibit signatures of "youth-associated microglia" and characteristics of cavity macrophages found in fluid niches such as the peritoneum. Our findings provide insights into IVM development and function, highlighting their therapeutic potential.

developmental biology↗

bioRxiv2025

Succinate Dehydrogenase-Deficient Cancer Cells Have Increased Susceptibility to Ym155 Induced DNA Damage

The hereditary pheochromocytoma and paraganglioma (hPPGL) syndrome is caused by inherited mutations in Succinate Dehydrogenase genes (SDHx). Affected individuals are predisposed to developing pheochromocytomas (Pheo), paragangliomas (PGL), renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GIST). Notably, tumors with succinate dehydrogenase subunit B (SDHB) deficiency demonstrate increased metastatic risk, for which treatments remain palliative. Hence, discovering novel therapeutic avenues to improve the prognosis for SDHB-cancer patients is an urgent need. Here we employed human SDHB-deficient UOK269 RCC cells (SDHB-KO) and isogenic SDHB-reconstituted control cells (SDHB-WT) to discover SDH-dependent mitochondria-directed cytotoxic agents. Given the reduced ATP-generating capacity of SDHB-KO cells, we hypothesized they would be uniquely sensitive to futile cycle induction with mitochondrial ionophores (2,4-Dinitrophenol (2-DNP), BAM15, Niclosamide, Nitazoxanide). Indeed, these compounds exhibited preferential cytotoxicity toward SDHB-KO cells. However, the chemotherapeutic compound Ym155 demonstrated the most potent and dramatic (five-fold) preferential cytotoxicity towards SDHB-KO cells. Importantly, the SDH-dependent cytotoxicity of Ym155 was validated in both primary human pheochromocytoma cells and mouse pheochromocytoma (MPC) cells. Furthermore, because few SDH-deficient cell lines are available, we buttressed our findings in additional relevant cell lines by modeling SDH-deficiency using chemical SDH enzyme inhibition with 3-nitropropionic acid (3-NPA). We observed a persistent cooperativity between SDH-deficiency and Ym155 cytotoxicity across multiple cell lineages and disease models. Mechanistically, Ym155-induced cytotoxicity was independent of its primary target, Survivin. Instead, SDH-deficiency sensitized cells to Ym155-induced DNA damage. Strikingly, the phenotype of SDH-deficient Ym155 sensitivity was recapitulated by inhibition of the histone demethylase KDM4, a downstream consequence of SDH deficiency. Thus, the accumulation of succinate in SDH-deficient tumors inhibits KDM4 activity, impairs DNA repair and yields enhanced susceptibility to Ym155-induced reactive oxygen species (ROS) generation. The identified intrinsic susceptibilities of SDHB-deficient cancers has the potential to be therapeutically leveraged.

cancer biology↗

bioRxiv2025

Therapeutic Potential of a Tectorigenin Derivative (TED) in Herpetic Stromal Keratitis: Antiviral and lmmunomodulatory Mechanisms

ObjectivesIn this study, the anti-HSV-1 effects of TED were investigated in vitro and in vivo, its therapeutic efficacy against herpetic stromal keratitis (HSK) was evaluated, and its mechanism of action was examined. MethodsWe assessed the cytotoxicity and anti-HSV-1 activity of TED in Vero cells using CCK-8 and plaque assays. The HSK model mice received TED (2.5, 5, or 15 mg/kg), and symptom progression was monitored. The therapeutic effects were evaluated through HE staining, corneal immunofluorescence, and HSV-1 gB quantification. Mechanistic studies were performed to examine the expression of TLR pathway genes, CD4+/CD8+ T-cell infiltration in the cornea and draining lymph nodes, and dendritic cell activation. ResultsTED showed potent anti-HSV-1 activity in vitro and ameliorated HSK in mice, improving body weight and reducing corneal pathology and the incidence of encephalitis. It decreased HSV-1 loads in the cornea, trigeminal ganglion, and brain while modulating cytokines (downregulating IFN-/{gamma}, TNF-, and IL-1{beta} and upregulating IL-10). TED suppressed the TLR pathway (TLR2/3/9, TRAF6, IRF3, and NF-{kappa}B) in the corneal epithelium and reduced CD4+/CD8+ T-cell and dendritic cell infiltration while increasing the number of CD4+ T cells in the lymph nodes. ConclusionsTED has significant anti-HSV-1 activity and effectively treats HSK by reducing viral loads, improving symptoms, and modulating immune responses through TLR pathway inhibition and immune cell regulation, suggesting its potential as an HSK therapeutic.

pharmacology and toxicology↗

bioRxiv2025

Continuous Serial Electron Diffraction for High Quality Protein Structures

Determining macromolecular structures is crucial for understanding biological mechanisms and advancing drug discovery. Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (MicroED) using continuous sample rotation has emerged as a powful method for solving structures from sub-micrometre-sized crystals. However, the resolution of MicroED data from protein crystals is often limited by radiation damage. Serial electron diffraction (SerialED) overcomes this limitation by merging single-shot diffraction patterns from thousands of crystals, but its widespread use has been hindered by the complexity and scarcity of equipment required for single shot data acquisition. Here, we introduce continuous SerialED (c-SerialED) - a simple, robust and widely accessible protocol. This approach collects diffraction data quickly and efficiently from all crystals within a given area, without prior crystal identification. We show that only using a standard cryo-EM instrument equipped with a simple widely available CMOS detector, c-SerialED greatly reduces radiation damage while improving the data quality. We demonstrate that c-SerialED enables determination of lysozyme structures at atomic resolution (0.83 [A]) and improves the data resolution of Dype Type Peroxidase Aa (DTPAa) crystals from 2.5 [A] (MicroED) to 1.3 [A]. Remarkably, the resulting structures are virtually free of radiation damage. The improved data quality and resolution allow visualization of radiation sensitive chemical features and protein-ligand interactions to state-of-the-art accuracy. By providing a convenient, fast, and damage-minimizing workflow on existing cryo-EM setups, c-SerialED significantly enhances the applicability of electron diffraction in structural biology. We anticipate our protocol will enable a wide range of studies requiring high-quality diffraction data from radiation-sensitive macromolecular crystals.

molecular biology↗

bioRxiv2025

A bacterial PrimPol-reverse transcriptase hybrid protein has a proofreading exonuclease activity that can be transferred to other reverse transcriptases

Gene disruption analysis revealed that an E. coli PPRT protein, which has an N-terminal Primase-Polymerase (PrimPol) domain fused to a group II intron-like reverse transcriptase (RT) domain followed by a long C-terminal domain (CTD), contributes to a cellular oxidative DNA damage response in addition to its previously described function in phage defense. Biochemical analysis showed that the PrimPol domain has an error-prone DNA polymerase activity that enables read through of oxidation-induced DNA damage. Surprisingly, we found that the RT-like domain, in addition to synthesizing protein-primed DNAs for phage defense, has a 3 to 5 DNA exonuclease activity that functions in proofreading DNAs synthesized by the PrimPol domain. Extending these findings, we identified structural features that contribute to this proofreading activity, enabling us to associate it with both a group II intron-encoded and retroviral RT and suggesting general methods for incorporating proofreading activity into RTs.

molecular biology↗

bioRxiv2025

Post-inhibitory rebound firing drives hypothalamic activation for male mating

Many behaviorally relevant limbic circuits are dominated by inhibitory connections, raising the question of how neuronal activation arises in such contexts. In male mating, both activation of mPOA and its inhibitory inputs are essential--a paradox previously ascribed to disinhibition. Here, we show that the mPOA largely lacks local inhibitory circuits, arguing against disinhibition. Instead, in vivo recordings reveal that mount-active mPOA neurons are suppressed prior to behavioral onset, and this inhibition negatively correlates with subsequent activation, consistent with post- inhibitory rebound firing (PIR). A biologically plausible conductance-based model demonstrates that synaptic inhibition interacts with T-type Ca{superscript 2} currents -- the ionic basis of PIR -- to promote mPOA neuron firing. Experimentally, bidirectional manipulations of mPOA T-type Ca{superscript 2} channels confirm this mechanism: knockdown reduces PIR and impairs male mating, while overexpression enhances mating. Together, these results identify PIR as a critical mechanism by which inhibition promotes activation in limbic circuits to drive male mating.

neuroscience↗

bioRxiv2025

MutagenesisForge: A Framework for Modeling Codon-Level Mutational Biases and Calculating dN/dS

The ratio of nonsynonymous (dN) to synonymous (dS) substitutions in protein-coding genes is a fundamental metric in molecular evolution to test hypotheses about the relative contributions of genetic drift and natural selection in shaping patterns of protein divergence (Williams et al., 2020). However, interpretation of dN/dS ratios may be confounded by sequence context and specific substitution models (Hughes, 2007; Kryazhimskiy & Plotkin, 2008). We present MutagenesisForge, a modular command-line tool and Python package for simulating codon-level mutagenesis and calculating dN/dS under user-specified conditions. At its core is the MutationModel interface which supports specific substitution matrices and ensures consistency across both Exhaustive and Contextual modes of simulation. These modes allow for users to test evolutionary hypotheses or to generate null distributions of dN/dS across a range of biologically relevant models. As large-scale DNA sequencing data sets continue to be generated both within and between species, MutagenesisForge offers a flexible platform for evolutionary analysis and hypothesis testing of mutational processes in protein-coding genes.

evolutionary biology↗

bioRxiv2025

IRE1α-XBP1s axis is required for BCG-induced trained immunity against viral infection by inhibiting AHR signaling

Although Bacille Calmette-Guerin (BCG) has been proved to be suitable in inducing trained immunity, mechanism-related issues have not yet been fully resolved. In this study, we found that in vitro BCG training protects both BCG harboring and bystander macrophages against DNA virus and RNA virus infection and demonstrated that BCG harboring cells protect bystanders partly by type I interferon (IFN). We determined aryl hydrocarbon receptor (AHR) as a negative regulator in BCG-induced trained immunity on defense against viral infection in vitro and in vivo and blockade of type I IFN signaling partially impaired the enhanced antiviral effect of AHR deficiency in BCG training. Furthermore, we revealed IRE1-XBP1s signaling as a critical pathway in BCG-induced trained immunity against viral infection in vitro and in mice. Mechanistically, we demonstrated that IRE1-XBP1s pathway influenced BCG-induced trained immunity by limiting AHR mRNA expression and the translocation of AHR protein into the nucleus.

immunology↗

bioRxiv2025

Telomere 3'-overhang attrition and persistent telomeric DNA damage response in failing human hearts

BackgroundTelomere homeostasis is critical for normal cellular and organ function, and its dysregulation is implicated in aging and chronic diseases. Although telomere length (TL) is critical for normal telomere function, its functional status can also be altered by many other factors. The organization and protective status of telomere in failing human heart (FHH) remains incompletely understood. MethodsUsing left ventricular tissues from patients with idiopathic dilated cardiomyopathy (IDC), ischemic heart disease (IHD), and non-failing heart (NFH), we performed a comprehensive analysis that included measurements of overall TL and 3 overhang length in left ventricles and cardiomyocytes (CM), assessment of telomere-binding protein associations, and transcriptomic profiling through RNA sequencing. ResultsAlthough TL varied among individuals, reduced median TL was observed only in IDC, while both IDC and IHD showed an increased frequency of very short telomeres, falling below the 5th percentile, in CMs. Strikingly, significant telomere 3-overhang attrition was detected in both disease groups and strongly correlated with elevated H2AX phosphorylated on serine 139 ({gamma}H2AX) in heart tissues, indicating DNA damage. This was accompanied by persistent activation of ataxia telangiectasia mutated (ATM) protein-mediated DNA damage responses and the formation of telomere dysfunction-induced foci (TIFs) in CMs from FHH. Concomitantly, the association of telomeres with the single-stranded telomere-binding protein, Protection of Telomeres 1 (POT1), and the double-stranded telomere-interacting protein, telomere repeat-binding factor 2 (TERF2), was markedly reduced, accompanied by an increase in association of {gamma}H2AX and ssDNA binding protein, RPA with telomeres in IDC and IHD relative to NFH, signifying telomere de-protection. ConclusionsCM telomere dysfunction, characterized by 3 overhang attrition and de-protection, is a common feature in FHH, leading to persistent DNA damage response in telomeres. Better understanding of telomere biology throughout the progression of different heart diseases to heart failure will provide more effective prevention and treatment strategies. GRAPHICAL ABSTRACTHuman telomeres safeguard chromosome ends through a higher-order telomere-loop (t-loop) structure and the shelterin complex, preventing their recognition as DNA double-strand breaks (DSBs). In the failing human heart (FHH) with idiopathic dilated cardiomyopathy (IDC) and ischemic heart disease (IHD), cardiomyocyte telomeres lose protection due to disruption of the shelterin complex, driven by oxidative DNA damage from reactive oxygen species (ROS) targeting G-rich telomeric sequences. This leads to t-loop unfolding, DDR cascade activation, 3 overhang excision, and persistent DNA damage responses with Telomere Dysfunction-Induced Foci (TIFs) formation at the telomeric regions of cardiomyocytes in FHH. The ATM-mediated DDR, along with engagement of both classical non-homologous end joining (cNHEJ) and homologous recombination (HDR) repair pathways, is integral to FHH progression. This study underscores the significance of telomere 3 overhang attrition in the pathogenesis of FHH with IDC and IHD. Unlike changes in overall telomere length, 3 overhang shortening directly correlates with increased DNA damage in human myocardial tissue. Preventing telomere 3 overhang attrition and oxidative damage offers a promising avenue for attenuating heart disease progression toward heart failure. In particular, selective ATM inhibition, as opposed to ATR inhibition, presents a potential therapeutic strategy for patients with IDC and IHD. Future efforts to pinpoint deficiencies in cNHEJ and HDR pathways in failing human hearts may reveal novel targets for heart failure prevention and treatment, providing new strategies in the management of this patient population. CMs, cardiomyocytes; DDR, DNA double-strand break repair; FHH, failing human heart; ROS, reactive oxygen species; t-loop, telomere-loop. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/669993v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1d21937org.highwire.dtl.DTLVardef@757f54org.highwire.dtl.DTLVardef@1fa7472org.highwire.dtl.DTLVardef@1f2e961_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Ultrasound-activated drug release with extracellular vesicles

Precise, noninvasive targeting and controlled drug release to the nervous system could transform both basic research and clinical therapy. In this work, we developed a focused ultrasound-responsive delivery system based on extracellular vesicles (EVs), which are naturally occurring and biocompatible phospholipid nanocarriers. To enable the ultrasound responsiveness, a lipid-gas phase transition material Perfluoropentane (PFP) was co-encapsulated with therapeutic agents into EVs via low-temperature sonication. The resulting EVs had a size of 100-200 nm and demonstrated efficient, on-demand release upon ultrasound stimulation. In primary cultured neurons, EVs loaded with lidocaine successfully suppressed calcium activity and showed minimum cytotoxicity. Finally, we demonstrated in vivo application of this system for modulation pain sensitivity in rats. Our EVs-drugs platform demonstrated a safe, and ultrasound-activated drug delivery with high spatiotemporal control, showing strong potential therapeutic applications.

bioengineering↗

bioRxiv2025

Comparison and Dynamic interaction between Auditory Cortex and Prefrontal Cortex of Behaving Monkeys during Novelty Detection

The ability to detect deviations from expected sensory input is fundamental for adaptive behavior. We recorded electrocorticographic activity from the auditory (AC) and prefrontal (PFC) cortices of behaving macaques during an auditory oddball task to probe the cortical dynamics of predictive processing. Repetition of standard stimuli evoked suppression and facilitation in AC and strong low-frequency (2 Hz) enhancement in PFC, accompanied by bidirectional delta-band coupling indicative of a shared predictive state. Deviant stimuli triggered early AC responses followed by PFC activation and increased feedforward and feedback connectivity across delta, theta, and gamma bands. Behavioral engagement amplified both prediction and prediction error signals, strengthening cortical network coordination. Together, these findings reveal a hierarchical predictive network in which the AC encodes sensory regularities and violations, while the PFC integrates predictive context in a behaviorally dependent manner.

neuroscience↗

bioRxiv2025

CircRNA-based, non-integrated, in vivo panCAR-mediated B cell immune resetting in mouse models and non-human primates

B cell immune resetting, defined as the comprehensive depletion and subsequent reconstitution of B cells, represents a pivotal therapeutic approach for B cell-associated disorders. Recently, chimeric antigen receptor T cell (CAR-T) therapy has demonstrated substantial clinical efficacy in treating B cell malignancies and autoimmune diseases. However, this approach remains constrained by its reliance on lymphodepleting conditioning regimens, high invasiveness, high costs, and the risk of severe cytokine release syndrome (CRS). Antibody-based therapies, including monoclonal antibodies and T cell-engager (TCE) bispecific antibodies, suffer from suboptimal tissue penetration, resulting in inadequate B cell depletion in lymphoid and peripheral tissues, and subsequent rapid resurgence of pathogenic B cells. Here, we engineered highly stable circular RNAs (circRNAs) encoding anti-CD19 CAR (circRNA-CAR), which demonstrated prolonged and enhanced CAR protein expression compared to conventional linear mRNAs. By employing immune cell-tropic lipid nanoparticles (LNPs) for in vivo delivery, circRNA-CAR facilitated the generation of panCAR immune cells (including CAR-T, CAR-NK, and CAR-macrophages) in both murine models and cynomolgus monkeys. Compared to rituximab or teclistamab, in vivo panCAR induced more extensive B cell depletion across multiple tissue compartments in mice. Moreover, in vivo panCAR mediated robust B cell depletion, eliminated autoreactive antibodies, and restored renal function by alleviating proteinuria in a systemic lupus erythematosus (SLE) murine model. It also effectively reduced IgE antibody levels, decreased eosinophil activity, and improved lung pathology in an asthma murine model. Notably, in vivo panCAR-mediated B cell depletion significantly attenuated aging-associated phenotypes in 17- month-old aging mice, remarkably outperforming both rituximab and teclistamab. In cynomolgus monkeys, in vivo panCAR achieved sustained B cell depletion lasting approximately 40 to 50 days, followed by reconstitution of a naive B cell repertoire. Collectively, circRNA-based in vivo panCAR enables complete B cell immune resetting, offering a versatile and promising therapeutic platform for a broad spectrum of B cell- and antibody-mediated disorders.

immunology↗

bioRxiv2025

Walking Selectively Modulates Behaviorally Relevant Auditory Responses According to Stride Cycle Phase

Perception in natural environments unfolds while the body is in motion, yet how locomotion shapes cortical computations for fine-grained and dynamic scene analysis remains unclear. Here, we combined over-ground walking with mobile EEG and a transitional click-train paradigm to test how natural locomotion influences auditory processing in humans. Participants listened to two-segment click trains in which a subtle inter-click-interval (ICI) change signaled a pitch shift and performed a task-engaged change-detection task while either sitting or walking; an independent cohort also completed a passive-listening version. Walking elevated behavioral detection thresholds and slowed responses, indicating reduced sensitivity to fine temporal changes. Neurally, locomotion produced a response-component-specific attenuation: activity time-locked to the ICI transition--the change response previously linked to temporally integrative updating of an ongoing auditory object--was strongly reduced, whereas the offset response was largely preserved and onset responses were only modestly affected. Critically, this modulation persisted during passive listening and was selectively amplified near the perceptual threshold during task-engaged detection, revealing an interaction between motor state and cognitive demand. Thus, natural locomotion does not uniformly degrade hearing; instead, it preferentially down-weights a behaviorally critical, temporally demanding stage of auditory processing, offering a framework for understanding how motor-cognitive state shapes auditory scene analysis in the moving brain.

neuroscience↗

bioRxiv2025

Interpretable spatial multi-omics data integration and dimension reduction with SpaMV

Spatial multi-omics technologies have revolutionized our understanding of biological systems by providing spatially resolved molecular profiles from multiple perspectives. Existing spatial multi-omics integration methods often assume that data from different modalities share a common underlying distribution, aiming to project them into a single unified latent space. This assumption, however, can obscure the unique insights offered by each modality, thereby limiting the full potential of multi-omics analyses. To address this limitation, we present the Spatial Multi-View (SpaMV) representation learning algorithm, which captures both the shared information across modalities and the distinct, modality-specific information, enabling a more comprehensive and interpretable representation of spatial multi-omics data. Through extensive evaluation on both simulated and real-world datasets, SpaMV demonstrates superior spatial domain clustering performance and provides users with more interpretable dimension reduction for downstream analysis. Moreover, SpaMV effectively annotates cell types within clusters of a mouse thymus dataset, highlighting its effectiveness in interpretable dimensionality reduction.

bioinformatics↗

bioRxiv2025

DNAH14 deficiency impairs sperm motility by reducing flagellar beat amplitude

Male infertility affects approximately 15-20% of couples worldwide, with asthenozoospermia accounting for 19% of cases. Nevertheless, the genetic basis of many asthenozoospermia cases remains poorly understood. In this study, we generated Dnah14 knockout mice, which displayed subfertility due to impaired sperm motility despite normal sperm morphology. Comprehensive kinematic analysis revealed that Dnah14-deficient spermatozoa exhibited reduced flagellar beat amplitude but increased beat frequency, while other dynein components remained unaffected. Notably, increasing sperm concentration during in vitro fertilization (IVF) experiments partially rescued the fertilization defects caused by Dnah14 deletion, suggesting a compensatory mechanism for this specific form of asthenozoospermia. Our results establish DNAH14 as a critical regulator of sperm motility and propose a potential therapeutic strategy for affected individuals.

developmental biology↗

bioRxiv2025

A stem cell-based platform for functional analysis of genetic variants in lung disease

Advances in genetic and transcriptomic technologies have identified large numbers of genes and variants of potential importance to human disease. Determining the function of these genes and variants is a critical bottleneck in understanding disease etiology. Variants of uncertain significance (VUS) are highly prevalent in our genomes, but our ability to identify them significantly outpaces our ability to determine their molecular and clinical consequences. We developed a genetically tractable induced pluripotent stem cell (iPSC) based platform to investigate gene variant pathogenicity in lung disease, using primary ciliary dyskinesia (PCD) as a model. We identified an individual with a clinical diagnosis of PCD and a VUS in the gene Multiciliate differentiation and DNA synthesis associated cell cycle protein (MCIDAS). Through gene-editing of iPSC-derived airway basal stem cells (iBCs), we precisely defined the molecular and cellular pathogenicity of the variant providing a successful application of the iPSC system to diagnose a lung disease.

cell biology↗

bioRxiv2025

Poison exon splicing in the human brain: a new frontier for understanding and targeting neurological disorders

BackgroundPoison exons (PE) are highly conserved exons whose inclusion creates premature termination codons (PTCs) and triggers nonsense-mediated decay (NMD) of the mature transcript. Despite their important role in post-transcriptional regulation, PEs remain poorly annotated due to the lack of systematic transcriptome-wide approaches. ResultsA comparative analysis of 957 eukaryotic transcriptomes revealed that Homo Sapiens exhibits the highest enrichment of NMD-targeted isoforms. By systematically predicting and annotating splicing events that generate PTCs, we found 9,814 PEs in the human genome. Using RNA-seq dataset from GTEx and BrainSpan, we analyzed PE splicing across tissues and developmental stages and identified 83 PEs uniquely found in the human brain, 973 PEs with brain-specific differential splicing, and 1,284 PEs differentially spliced during brain development. By integrating ClinVar variant annotations with SpliceAI predictions, we identified 1,625 pathogenic variants associated with neurological disorders that are predicted to affect the splicing of 743 PEs, and we functionally validated the impact of a subset of them using CRISPR prime-editing in human cells. Functional characterization of the deep intronic NDUFAF6 c.420+784C>T variant associated with Leigh syndrome demonstrated increased PE inclusion and a significant reduction in NDUFAF6 protein levels in iPSC-derived induced neurons. Mutant cells also exhibited impaired mitochondrial membrane potential compared to controls. Together, these findings provide functional evidence that the c.420+784C>T variant promotes aberrant PE inclusion, leading to reduced NDUFAF6 expression and mitochondrial dysfunction. ConclusionsOur findings highlight PEs as pivotal regulators of gene expression in the human brain and support the therapeutic targeting of PE splicing in neurological diseases.

genetics↗