Biology subjects

Lee, E.

Publications and source records attributed to Lee, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Developmental origins of thymic B-cell lymphomas

The thymus provides a specialized niche for lymphocyte differentiation, yet the developmental trajectories of its rare B-cell population and their relationship to thymic B-cell lymphomagenesis remain poorly understood. Here, we integrate single-cell multiomics and spatial profiling across human tissues to delineate a B-cell differentiation program selectively represented in the human thymus. This program links CCL17 and germinal center-like B-cell states along a trajectory distinct from canonical germinal center differentiation and can be recapitulated in vitro by signals operating in the thymic medulla. Extending this framework across human B-cell lymphomas, we identify an inherent malignant cell state in PMBCL that shares transcriptional and epigenetic features with CCL17 thymic B cells. This malignant state associates with early clonal positions in reconstructed B-cell receptor phylogenies and persists within tumor microenvironments distinct from the thymic niche. Together, these findings support a model in which thymus-specific B-cell differentiation programs are redeployed during thymic B-cell lymphomagenesis.

cancer biology↗

bioRxiv2026

Brainstem premotor circuits transform dental sensory input into coordinated jaw opening

Protective actions require the rapid transformation of damage into coordinated activity of motor pools, yet the brainstem circuits implementing this operation remain poorly defined. Here, we use the tooth-trigeminal system to identify molecularly specified pathways linking intradental sensory input to opposing jaw motor pools. Optogenetic activation of tooth-associated sensory afferents evoked rapid, stimulus-locked jaw-opening. Intradental afferents formed functional monosynaptic connections with Pdyn+ neurons in the 5Sol transition zone and Calb2+ neurons in the 5Tr transition zone, identifying these regions as sensory-recipient premotor interfaces. Predominantly excitatory Pdyn+ 5Sol neurons provided monosynaptic input to anterior digastric motor neurons, whereas predominantly inhibitory Calb2+ 5Tr neurons provided bilateral monosynaptic input to masseter motor neurons. Activation of Pdyn+ 5Sol neurons was sufficient to evoke jaw opening. Together, these findings define complementary brainstem pathways positioned to couple jaw-opening drive with inhibition of antagonistic jaw-closing output, revealing a withdrawal-like sensorimotor computation adapted to cranial biomechanics.

neuroscience↗

bioRxiv2026

CalFluxTools: An R package for analysis of high-throughput calcium oscillation screening data

Whole plate detection of calcium flux signals using fluorescence is a powerful technology for monitoring oscillatory activity of excitable cells in a high throughput fashion that sees broad applications in neurotoxicity and cardiotoxicity research. However, analysis of calcium oscillation profiles can output more than 60 peak kinetic parameters, which makes manual data analysis challenging. We have created the CalFluxTools R package, an automated pipeline for parsing plate data for 384 well plates, generating quality control metrics and plots, and performing data analysis, including T-tests, Z-score analysis, and machine learning prediction of compound toxicity values. CalFluxTools requires a plate map and experimental manifest that includes parameter filtration specifications, as well as data in the ".statall" format. CalFluxTools runs in less than a minute on a Macbook Pro with a 2.6 GHz 6-Core Intel Core i7 processor.

bioinformatics↗

bioRxiv2026

Characterizing the interaction of a type VII-secreted antimycobacterial toxin with its small helical partner proteins

The type VII secretion system (T7SS) is a membrane-embedded protein export pathway found in mycobacteria and Gram-positive bacteria. Recently it was shown that Mycobacterium abscessus uses its ESX-4 variant of the T7SS to secrete a toxin, EatA, which targets arabinogalactan present in the mycobacterial cell envelope. Prior to its export, EatA forms a complex with a pair of small proteins from the WXG100 family, TapA1 and TapA2. Here we investigated a structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 using site-directed mutagenesis and bacterial 2-hybrid assays. Our results are consistent with the three proteins forming a stacked bundle of alpha-helices. Structural modelling also predicted an interaction of the EatA-TapA1-TapA2 complex with EsxT-EsxU, a second pair of WXG100-family proteins that are likely required for the mechanistic operation of ESX-4. Whilst we could demonstrate a potential interaction between TapA2 and EsxT by bacterial 2-hybrid analysis, we were not able to purify a complex of all five proteins.

microbiology↗

bioRxiv2026

Single-cell spatial multi-omic characterization of the tumour microenvironment in transformed follicular lymphoma

Histological examination of follicular lymphoma (FL) biopsies remains the cornerstone for diagnostic grading of FL. Single-cell sequencing approaches, while transcriptomically rich, require tissue dissociation and lose the native spatial context that underpins FL transformation to diffuse large B-cell lymphoma (DLBCL). To investigate the spatial interplay between malignant B-cells and the tumour microenvironment (TME) across disease states, we performed subcellular single-cell spatial transcriptomics and spatial proteomics on 12 paired pre/post-transformation samples and 10 non-transforming FL controls, integrated with matched single-cell whole genome sequencing (scWGS). Our analysis reveals that transformation is accompanied by a shift toward B-cell-predominant stromal and immunosuppressive cellular neighbourhoods, where the magnitude of expansion correlates with time to transformation. Prior to transformation, immunomodulatory Galectin-9 interactions move from the intra-follicular core to the extra-follicular space. Integration with scWGS demonstrates that high copy-number instability in malignant B-cells is associated with reduced supportive T-cell niches and intensified immunoregulatory crosstalk at the transformed state. Collectively, our multi-omic analysis characterizes TME remodeling during FL transformation, contributing to a refined disease evolution model.

bioinformatics↗

bioRxiv2026

Investigating Invariances in Auditory Event Categorization with Model Metamers

Real-world acoustic inputs contain rich sensory information that we parse into discrete auditory objects and categories. Although deep neural networks (DNNs) are increasingly used to model auditory perception, the field lacks rigorous behavioral benchmarks, particularly for auditory event categorization. Here, we developed a 25-way categorization paradigm for broad classes of natural sounds to test whether categorical invariances of DNNs align with those of human observers. We first confirmed that humans could reliably categorize the natural sounds, demonstrating that our paradigm is well-suited for testing invariances in auditory categories. To probe model invariances, we evaluated human recognition of model metamers (synthetic stimuli matched to the models internal activations for each natural stimulus) for a wide range of architectures trained on speech or auditory event recognition. Evaluating widely-used public models, we found that human recognition of auditory event model metamers was generally influenced by the training task and data distribution; speech models trained on standard, curated datasets produced less recognizable metamers than auditory event recognition models. We additionally analyzed a controlled set of models to directly investigate the influence of training task and adversarial training, revealing that improved metamer recognition induced by adversarial training is task-dependent. However, even in the best-performing models, we observed a sharp decline in human recognition at the final classification layer compared to the penultimate representation layer. Overall, our results suggest that while invariances in modern architectures better align with human observers for auditory event categorization, there is still a large discrepancy between the categorical invariances of auditory neural networks and the invariances of human observers. Code and models are available at https://github.com/Feather-Lab/env-sound-metamers

neuroscience↗

bioRxiv2026

S100A9-Dependent CXCR2hi Neutrophils Mediate Systemic Immune Suppression and Checkpoint Resistance in Metastatic TNBC

Most high-dimensional studies of tumor-immune interactions focus on metastatic models, limiting insight into how immune remodeling in primary tumors shapes metastatic competence. Here, integrating single-cell RNA sequencing, CyTOF, and functional studies across metastatic (4T1) and non-invasive (EMT6) triple-negative breast cancer (TNBC) murine models, we define tumor state-specific immune programs that distinguish metastatic competence. Tumors with metastatic capacity uniquely drive early bone marrow expansion of CXCR2 neutrophils, which infiltrate primary tumors acquiring a CXCL2-producing phenotype that promotes EMT-associated cancer stem cell (CSC) plasticity. This program depends on TGF-{beta}/CEBPD-mediated induction of S100A9. Elevated CXCL2, together with G-CSF, establishes a feed-forward circuit that drives systemic neutrophil mobilization and recruitment to distant organs, where neutrophil-derived S100A8/A9 (calprotectin) promotes MET-driven CSC outgrowth and metastatic colonization. Clinically, gene signatures associated with CXCR2 neutrophils predict poor survival in TNBC patients, whereas monocyte/macrophage (CX3CR1) and T cell activation signatures correlate with improved outcomes. S100A9 ablation disrupts this cascade and enhances immunotherapy responsiveness, defining a TGF-{beta}/S100A9/CXCR2 axis linking immune remodeling, CSC plasticity and metastasis. HighlightsO_LIMetastatic TNBC engages a TGF-{beta}/C/EBP{delta}/S100A9 axis that expands CXCR2 neutrophils C_LIO_LINon-invasive EMT6 tumors retain a CX3CR1 monocyte/macrophage and T-cell landscape C_LIO_LICXCR2+ neutrophils in pre-metastatic niches suppress T cell response while promoting tumor cell proliferation C_LIO_LIS100A9 loss redirects myelopoiesis and potentiates anti-PD-L1 in TNBC models C_LI In BriefAlkan et al. dissect how tumor state programs the myeloid compartment in TNBC. Metastatic 4T1 tumors uniquely engage a TGF-{beta}/C/EBP{delta}/S100A9 axis driving CXCR2 neutrophil expansion and CXCL2/G-CSF-dependent systemic mobilization, coupling immune remodeling to EMT/MET cancer-stem-cell plasticity, while S100A9 loss restores CX3CR1 myeloid identity and unlocks checkpoint-inhibitor responsiveness.

cancer biology↗

bioRxiv2026

Competition against the intestinal microbiota selects for pathoadaptive traits in ESBL E. coli

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

microbiology↗

bioRxiv2026

Rapid vaccine induction of macaque HIV-1 V2 Apex broadly neutralizing antibodies with immunogenetic signatures that are potentially translatable to humans

Due to the complex biology of HIV-1 broadly neutralizing antibodies (bnAbs), HIV-1 vaccines must target unmutated germline precursor antibodies with B cell receptors that have the appropriate specificity and genetic features to develop neutralization breadth. Previous work has shown that immunization or infection in macaques primes unmutated precursor antibodies that target the broadly neutralizing epitope composed of the second variable region and proximal glycans (termed V2-apex site) of HIV-1 envelope (Env). Macaque V2-apex bnAb precursors elicited thus far have homogeneous usage of the second reading frame of a diversity (D) gene found only in macaques, calling into question whether HIV-1 Env immunogens can elicit only this canonical, macaque-specific V2-apex antibody response, limiting human vaccination success. Here, we show that vaccination of rhesus macaques with Env conjugated to a ferritin nanoparticle (OPT4-scNP) rapidly elicited V2-apex bnAbs with distinct D gene segment usage and motifs that are not restricted only to macaques. After two priming immunizations, V2-apex antibodies with non-canonical HCDR3s exhibited greater neutralization potency and breadth than V2-apex antibodies with previously observed HCDR3 motifs. Following sequential boosting immunizations, a V2-apex bnAb lineage with a novel HCDR3-encoded EDGED motif increased its neutralization breadth, including neutralization of viral isolates bearing the N130 glycan, which shields the V2-apex from recognition. High-resolution structures of two members of this V2-apex bnAb lineage, called DH2050, defined a novel binding mode in which it used its EDGED motif to contact the C-strand peptide and the N160 glycan within the V2-apex site. Altogether, we define a novel bnAb binding mode to the V2 Apex and demonstrate that HIV-1 Env sequential vaccination elicits V2-apex bnAbs with immunogenetic signatures that are potentially translatable to the human immune system.

immunology↗

bioRxiv2026

Cutaneous inflammation accelerates the premalignant expansion of melanocytes bearing oncogenic mutations

How the cutaneous microenvironment influences early melanomagenesis is poorly understood. Here, we assessed the effects of three immune perturbations on premalignant melanocyte expansion in an autochthonous mouse model of disease. Depletion of regulatory T (Treg) cells markedly accelerated melanoproliferation, an unexpected phenotype that was associated with monocyte and macrophage infiltration, the production of inflammatory and angiogenic factors, and vascular leakage. In line with these observations, single cell transcriptomic analysis of Treg cell deficient skin revealed robust accumulation of monocyte-derived macrophages with tissue remodeling characteristics. Acute UV irradiation and 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity had analogous effects on both the cellular microenvironment of the skin and the expansion of local premalignant melanocytes. Treatment with the anti-inflammatory agent dexamethasone attenuated DNFB-induced melanocyte expansion and vascular remodeling. Collectively, these results identify a conserved inflammatory axis linked to the early outgrowth of oncogenic melanocytes in the skin.

cancer biology↗

bioRxiv2026

A Novel Network Approach to Identify Sample-Specific Context-Informed Metabolic Signatures During Developmental Processes

Metabolism plays an essential role in cellular processes: development, growth, differentiation, and determination of cell identity. Understanding how metabolic processes dynamically change across cell types, stages, and environmental conditions is crucial for studying developmental biology, aging, and disease progression. Genome-wide metabolic models (GEMs) are a powerful network-based tool for studying these processes by integrating omics data to model context-specific metabolism. However, current approaches, such as Flux Balance Analysis (FBA), have limitations in addressing the dynamic nature of metabolism across developmental stages at a sample-specific resolution. To address this, we introduce a novel network-based method for analyzing cell and stage specific metabolic flow using directed and weighted metabolic networks that account for sample-specific transcriptomic data. We apply this method to study ovarian follicle development, providing a deeper understanding of intra-cellular metabolic processes, identifying key metabolites, enzymes, and potential markers for follicular maturation, important for IVF. By incorporating biologically meaningful data, this approach bridges the gap between theoretical metabolic network models (GEMs) and experimental observations, offering a systems-level view of metabolic dynamics in developmental and understudied contexts.

bioinformatics↗

bioRxiv2026

Subclonal IDH1/2 Mutations as a Targetable Vulnerability in Vascular Tumors

Despite extensive sequencing, the genetic etiology of sporadic angiosarcoma remains poorly defined (1-3). Maffucci syndrome, characterized by vascular tumors and elevated cancer risk, is driven by mosaic gain-of-function mutations in IDH1/2 (4,5), though these have not been reported in sporadic angiosarcoma. We identify recurrent, low-variant allele frequency hotspot mutations in IDH1/2 in over half of sporadic angiosarcomas. Mutations were validated by Sanger sequencing and immunohistochemistry. Mutant IDH1 endothelial cells promote tumorigenesis through non-cell-autonomous mechanisms, secreting 2-hydroxyglutarate (2-HG) to increase growth factor and endothelial-to-mesenchymal transition gene expression, activate pAkt/pERK signaling, induce DNA methylation changes, and promote anchorage-independent growth, which are reversed by the mutant IDH1 inhibitor ivosidenib. Patients with mosaic IDH1 mutations show reduced serum 2-HG and marked tumor regression following ivosidenib treatment. The clinical efficacy of ivosidenib in vascular tumors with subclonal IDH1 mutations suggests that low VAF IDH1/2 mutations may be a targetable vulnerability in sporadic angiosarcoma. (6,7) Statement of SignificanceWe identify recurrent, low-VAF IDH1/2 mutations in angiosarcoma and provide evidence that these subclonal mutations promote tumorigenesis through non-cell-autonomous mechanisms. Vascular tumors driven by subclonal IDH1 mutations responded dramatically to ivosidenib, thus revealing a novel treatment for a subset of vascular tumors.

cancer biology↗

bioRxiv2026

Discovery of a CI-994 derivative as a dual modulator of class I HDACs and Wnt/β-catenin signaling for Alzheimer's disease therapy

Alzheimers disease (AD) is a multifactorial disease with mixed pathologies. Consequentially, drugs targeting multiple pathological processes may offer synergistic benefits. While histone deacetylase (HDAC) inhibitors have demonstrated efficacy in alleviating AD-related pathologies in animal models, the neuroprotective Wnt/{beta}-catenin signaling pathway remains compromised in AD brain. CI-994 is a class I HDAC inhibitor containing N-(2-aminophenyl)-benzamide. Our recent studies indicate that CI-994 is also an activator of Wnt/{beta}-catenin signaling by stabilizing Wnt co-receptor LRP6. We herein use CI-994 as a scaffold to develop novel potent dual modulators of class I HDACs and Wnt/{beta}-catenin signaling for AD therapy. Our lead compound, W2A-28, selectively inhibits class I HDAC1, 2 and 3 with IC50 values of 0.51 M, 0.68 M, and 0.22 M, respectively, and shows no inhibitory activities on other HDACs. Furthermore, W2A-28 potently activates Wnt reporter activity with an EC50 value of 1.61 M in Wnt-3A-expressing HEK293 cells. As expected, activation of Wnt/{beta}-catenin signaling by W2A-28 is associated with elevated LRP6 protein level. Importantly, W2A-28 displays excellent microsomal stability in both mouse and human liver microsomal stability assays, alongside high permeability and a lack of active efflux in MDR1-MDCKII models. Critically, W2A-28 treatment significantly enhances histone acetylation, activates Wnt/{beta}-catenin signaling, and suppresses tau phosphorylation in AD patient-specific cerebral organoids carrying APOE {varepsilon}4/{varepsilon}4 or APOE {varepsilon}3/{varepsilon}4 with PSEN1 M146V mutation. Our findings position W2A-28 as a promising multi-target drug candidate for AD therapy.

neuroscience↗

bioRxiv2026

Molecular Logic of DNMT3A1 Recruitment: Resolving Multivalency at the Chromatin Interface

Histone modifications correlate with DNA methylation, but the underlying mechanisms remain unclear. The DNA methyltransferase DNMT3A1 engages nucleosomes through multivalent interactions with linker DNA, histone H3 tails, the acidic patch, and ubiquitinated H2A. Using binding assays, kinetic analyses, and nanopore sequencing of engineered nucleosomes, we show that DNMT3A1 binding reflects avidity, limiting the impact of individual contacts. Accordingly, disrupting single interactions minimally affects overall affinity, whereas isolated domains and truncations remain modification-sensitive. Although histone modifications have little effect on kcat/Km for linker methylation, disrupting ADD-H3K4me0 interactions redistributes methylation away from the nucleosome core particle. Nucleosome competition assays reveal that H3K4me0 and H3K36me2 promote selective linker methylation, whereas H3K27me3 and PRC2/EZH2 have no effect. Notably, despite strong UDR-dependent binding to H2AK119ub1-modified nucleosomes, this mark fails to enhance methylation over an unmodified competitor. We propose a model of commitment to catalysis to reconcile weak kinetic differences with strong substrate selectivity. These findings highlight avidity and commitment in governing DNMT3A1 nucleosome recognition and DNA methylation specificity.

molecular biology↗

bioRxiv2026

Agent-Guided De Novo Design of Nanobody Binders Against a Novel Cancer Target

Therapeutic antibody discovery remains slow and resource-intensive, with traditional methods providing limited control over epitope selection. We present a workflow for de novo nanobody design applied to a novel Desmoplastic Small Round Cell Tumor target encompassing four stages: (1) epitope identification guided by our hotspot recommendation agent using physical chemistry-based structure and sequence analysis tools with two curated databases (IEDB, PFAM), (2) de novo nanobody generation using three independent methods (RFantibody, IgGM, mBER) across multiple predicted antigen structures and nanobody frameworks, (3) multi-metric scoring including structural metrics from folding models, and in silico binding affinity from our sequence-based predictor, (4) high-throughput yeast surface display (YSD) screening followed by surface plasmon resonance (SPR) characterization of the specific binders. We generated 288,000 nanobody designs spanning eight target epitope regions and three variable domains of heavy chain-only antibody (VHH) frameworks. Multi-objective Pareto filtering with our candidate selection agent yielded 100,000 candidates for YSD screening with fluorescence-activated cell sorting (FACS). Of 116 enriched candidates advanced to SPR characterization, 46/116 (39.7%) produced reliable kinetic fits with Rmax [≥] 30 RU, yielding KD values from 0.66 nM to 305 nM (median 31.7 nM). These results show that an agent-guided computational workflow can design nanomolar to sub-nanomolar nanobody binders against a novel target without experimental structure or prior antibody information.

bioinformatics↗

bioRxiv2026

A Scalable fMRI Estimate of Basal Ganglia Brain Tissue Iron for Use in Developmental and Translational Neuroscience

Dopaminergic (DA) function and basal ganglia neurobiology are central to reward learning, motivation, and cognitive control, and dysregulation of these systems contributes to neuropsychiatric conditions that emerge during development. Adolescence is marked by profound reorganization of DAergic basal ganglia circuitry, yet direct in vivo assessment of the DA system remains limited in youth. Brain tissue iron is a developmentally sensitive marker of DA-related neurobiology that can be measured non-invasively via magnetic resonance imaging (MRI). Iron is an essential co-factor for DA synthesis and a foundational metabolic resource that supports cellular metabolism, myelination, and energetic demands of the basal ganglia. T2*-weighted echo-planar imaging (EPI), collected during functional MRI (fMRI), is sensitive to magnetic susceptibility of non-heme brain iron. Leveraging this property, we demonstrate the validity and broad applicability of an iron-sensitive metric that can be derived from conventional single-echo fMRI: {Delta}R2*. In a longitudinal developmental dataset (N = 151; age range 12-31), {Delta}R2* showed high reliability, strong longitudinal stability, and validity via robust convergence with established quantitative relaxometry-based iron measures (R2* and R2). Critically, {Delta}R2* can be retrospectively estimated from extant fMRI data and derived in large-scale consortium data repositories, demonstrated here in the Adolescent Brain and Cognitive Development (ABCD) baseline cohort (N = 8,366; ages 9-11). We show that {Delta}R2* captures known age-related increases in basal ganglia iron, highlighting neurodevelopmental sensitivity at population-scale. Together, these findings establish {Delta}R2* as a reliable, widely accessible marker of basal ganglia iron, enabling scalable investigation of lifespan trajectories and neuropsychiatric risk in existing and future datasets.

neuroscience↗

bioRxiv2026

SIRT5 acts in the tumor microenvironment via endothelial cell metabolism to support breast cancer growth

Triple-negative breast cancer (TNBC) is characterized by aggressive progression and poor prognosis, partly due to abnormal angiogenesis. While the metabolic reprogramming of tumor cells is well characterized, the metabolic regulation of tumor-associated endothelial cells (ECs) remains unclear. Here, we identified the mitochondrial deacylase SIRT5, which has established tumor-promoting roles in TNBC cells, as a key regulator of endothelial metabolic homeostasis and tumor angiogenesis. SIRT5-deficient host mice showed significant defects in supporting the growth of orthotopic SIRT5-proficient mammary tumor transplants, and the resulting neoplasms showed defects in tumor vascularization. In a 3D microfluidic vessel-on-chip model, SIRT5 loss compromised vascular barrier integrity and EC sprouting. Mechanistically, SIRT5-deficient ECs exhibited diminished mitochondrial respiratory capacity but apparently normal glycolysis. SIRT5 loss also caused increased mitochondrial reactive oxygen species levels, and a mitochondrial antioxidant rescued the endothelial cell defects following SIRT5 loss, indicating that SIRT5-mediated mitochondrial redox homeostasis in the tumor microenvironment is necessary to maintain vascular function. Orthotopic co-transplantation of TNBC and EC cells with or without SIRT5 knockdown demonstrated that endothelial SIRT5 promotes increased tumor growth in vivo. These results suggest that targeting SIRT5 offers a potential therapeutic strategy to disrupt tumor angiogenesis and suppress TNBC progression by targeting the metabolic vulnerabilities of the tumor endothelium.

cancer biology↗

bioRxiv2026

Regulation and Function of the HPV16 CircE7 RNA

High-risk human papillomaviruses (HPV), including HPV16, produce circular RNA that encompass the E7 oncogene (circE7). CircE7 can be detected in HPV16-positive cells and tumors, is preferentially localized to the cytoplasm, is N6-methyladenosine (m6A) modified and can be translated to produce the E7 oncoprotein. Here, we explored the regulation and function of circE7. Mutation of m6A motifs flanking the backsplice junction revealed a single essential m6A motif to be essential for circE7 formation. Mutation of this m6A motif promoted linear splicing of the E6*I splice site (226^409), suggesting that linear and circular E7 splicing are inversely regulated. Additionally, mutation of an IRES-like motif in circE7 significantly decreased E7 protein expression, without having significant effects on circE7 RNA levels. Knockdown of YTHDC1, but not other m6A-binding proteins, decreased both circE7 RNA and protein expression. BaseScope ISH was used to confirm the expression of circE7 in HPV positive head and neck squamous cell carcinoma cell lines. Using both qRT-PCR and BaseScope ISH, we found that serum and amino acid starvation significantly increased circE7 levels. Finally, we generated a HPV16 genome with two point mutations in the circE7 m6A motif (Mut2). Stable transduction of primary keratinocytes with Mut2 confirmed the loss of circE7 and increased expression of E6*I. The Mut2 HPV16 genome exhibited significantly decreased viral replication but an increased ability to transform primary keratinocytes. Our studies reveal that the precise regulation of circE7 and E6*I by m6A are critical for the ability of HPV16 to infect and transform keratinocytes.

molecular biology↗