Biology subjects

Murphy, S.

Publications and source records attributed to Murphy, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Inhibition of CDK7, CDK9 or CDK12 affects phosphorylation of human SPT5.

SPT5, complexed with SPT4 in the DRB-sensitivity-inducing factor, DSIF, is a critical regulator of transcription elongation that acts negatively in the unphosphorylated form and positively when phosphorylated. In vivo, phosphorylation of multiple sites in the largely unstructured linker and C-terminal regions of the protein facilitates release from an early elongation checkpoint and processive transcription elongation. Transcription elongation factors, including the PAF complex (PAF1C), interact with phosphorylated SPT5 and phosphorylation of residues S666 and T806 are associated with the positive elongation functions of SPT5. Here, using in vitro phosphorylation of regions of the SPT5 C-terminus followed by GST-mediated pull-down we confirm that the RTF1 subunit of PAF1C interacts with residues 750-850 only when phosphorylated. Antibodies we raised to phospho-S666 or phospho-T806 are specific for these sites phosphorylated in vitro and in vivo. Western analysis of endogenous SPT5 in HEK293 cells using these antibodies before and after inhibition of analog-sensitive versions of CDK7, CDK9 and CDK12 indicate that all three kinases play roles in the phosphorylation of SPT5, although inhibition of CDK9 has the most marked effect. CDK9 inhibition also inhibits the association of PAF1C subunits with SPT5 in vivo and affects phosphorylation of chromatin-associated SPT5 as measured by chromatin immunoprecipitation (ChIP).

molecular biology↗

bioRxiv2026

Two decades of Novel Ecosystems: Literature analysis of topics, trends and connectivity

1. As human-driven climate change and other anthropogenic forces produce widespread impacts on biodiversity and ecosystem functioning, many ecosystems are shifting into unprecedented, novel states. The Novel Ecosystem Concept (NEC), formalized in 2006, was developed as an alternative framework for managing "novel ecosystems", human-altered systems that are challenging to restore to their historical state. In the years since, the NEC has been proposed as a broader lens for ecosystem management under ongoing global change. 2. We evaluated how the uptake and potential of the NEC have been realized over time using a bibliometric and linguistic toolkit. Using bibliometric data from the Web of Science, we clustered 40,914 applied ecology papers published between 2006 and 2025 into twenty research topics based on language use. We quantified the volume of NEC research within each topic, rates of publication over time, and cross-topic connectivity proxied by citations. 3. We found NEC research in most of the topics of applied ecology, particularly invasion biology, ecosystem services, soil research and studies of freshwater, urban and marine systems. In contrast, several major conservation-focused topics, including protected areas, threatened species, and human-wildlife interactions, contained little research with an explicit link to novel ecosystems. Publication rates increased across all topics, although growth slowed over time. At the same time, NEC research became more focused into ecosystem-specific topics, especially marine ecosystems. NEC research across different topics was well-connected by citations, with papers more likely to cite each other when they used similar language. 4. Overall, the NEC does not appear to have expanded beyond its origins in restoration to become a broadly applied framework across conservation and ecosystem management. Instead, the concepts behind the NEC have been integrated into restoration and management frameworks grappling with climate and other anthropogenic change, while "novelty" is emerging as a powerful quantitative variable for measuring ecological change over space and time. Our results highlight the potential value of bibliometric and linguistic tools to deconstruct the ecological literature to inform what we can, or should, do to intervene for benefit of nature and people.

ecology↗

bioRxiv2026

CMAC: A deep learning framework for absolute cardiomyocyte transcriptional age estimation

Cardiomyocyte maturation spans embryogenesis through postnatal life and involves coordinated transcriptional transitions that are disrupted in disease and incompletely recapitulated by stem cell-derived cardiomyocytes. However, no unified framework exists for assigning absolute transcriptional maturation age across datasets, platforms, and experimental conditions. Here, we developed CMAC (Cardiomyocyte Maturation Age Clock), integrating 18 murine single-cell RNA-sequencing datasets spanning embryonic and postnatal timepoints, into a batch-invariant probabilistic atlas using single cell variational inference (scVI) with a 100-dimensional latent representation. Analysis of the developmental trajectory revealed highly non-uniform transcriptional change, with 63.9% of the total transcriptional journey completed by birth and the neonatal transition representing the largest single transcriptional step. An XGBoost regressor trained on the CMAC latent space predicted absolute chronological age with a leave-one-dataset-out mean absolute error (MAE) of 4.09 days (median 3.27 days), Pearson r = 0.903, and Spearman {rho} = 0.908 across 18 datasets and six sequencing platforms, outperforming linear regression (MAE = 6.18 days) and PCA-based representations (MAE = 6.35 days). In an independent P14 cohort comprising 98,163 cardiomyocytes from six biological replicates, CMAC achieved a mouse-level MAE of 0.33 days, with all six mice predicted within 1.1 days of chronological age. Application of CMAC to a previously published PGC1/{beta} double-knockout model with established delayed cardiomyocyte maturation independently recapitulated this phenotype, quantifying maturation deficits of 4.77 to 8.60 days across P7 to P28, with the largest deficit at P14. Together, these results establish CMAC as a quantitative, deep learning framework for assigning absolute transcriptional maturation age and quantifying deviations from normal cardiomyocyte development.

genomics↗

bioRxiv2026

Sex-divergent trajectories of hippocampal and cortical NMDA receptor density across the Alzheimer 's disease continuum

While loss of NMDA receptors (NMDARs) is associated with Alzheimers disease (AD) severity, the effect of sex or the relationship between regional NMDAR density and antemortem cognitive status across the AD spectrum has not been examined. We performed quantitative in vitro autoradiography of hippocampus, entorhinal cortex (EC), and parietal cortex using NMDAR and tau radioligands. Relationships between regional NMDAR density and cognitive status assessed by the Mini Mental State Exam (MMSE), and between NMDAR and tau density, were examined by bivariate correlations. In both sexes, the largest AD-related decreases in NMDAR density were observed in the CA1 field. However, there was a significant diagnosis by sex interaction driven by sex-specific changes in the mild cognitive impairment (MCI) stage, with lower NMDAR density in MCI women, but not MCI men relative to same-sex controls. Within diagnosis analyses revealed positive correlations between NMDAR density and MMSE scores and significant negative correlations between EC NMDAR and tau density, which was significant only in AD men. Our data show that changes in hippocampal NMDAR density across the AD continuum are modulated by sex and may contribute to the known sex differences in the clinical trajectory of the disease.

neuroscience↗

bioRxiv2026

Splicing Factor SF3B2 Regulates Cardiomyocyte Calcium Handling Through Alternative Splicing of Cardiac Ion Channel Genes

BackgroundAlternative splicing is a critical determinant of protein diversity in the heart, where it drives the postnatal functional maturation of cardiomyocytes and specifies the ion-channel and calcium-handling isoforms required for mature contractile function; dysregulated splicing programs have in turn been implicated in cardiomyopathies and arrhythmias. However, the splicing regulators that control cardiomyocyte calcium handling remain largely unknown. ObjectiveWe systematically screened 276 splicing factor genes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to identify regulators of calcium handling, and we selected SF3B2, a core U2 snRNP spliceosome component, for detailed follow-up based on complex-level enrichment and prior identification by Murphy et al. 2021 [1]. MethodsA high-throughput siRNA screen targeting 276 splicing factor genes was performed in hiPSC-CMs using 384-well calcium transient imaging. SF3B2 knockdown was followed by deep bulk RNA-seq analysis (n = 3 per group) for differential gene expression with DESeq2 and alternative splicing quantification with rMATS. RNA immunoprecipitation sequencing (RIP-seq) using an epitope-tagged SF3B2 construct was performed to identify direct mRNA binding targets of SF3B2. ResultsThe screen identified multiple U2 snRNP components, including SF3A2, SF3B1, and SF3B4--as regulators of calcium transient duration. SF3B2 was previously identified as a regulator of contractility in this screen [1], and the enrichment of its complex partners above the significance threshold in the current CTD75 analysis supported its selection for follow-up characterization. SF3B2 knockdown resulted in 2,683 differentially expressed genes (adjusted p < 0.05), with downregulated genes enriched in cell cycle and DNA replication pathways. Alternative splicing analysis revealed significant changes across all five rMATS event types in 36 genes within the cardiac muscle cell action potential involved in contraction gene ontology term (GO:0086002), including calcium channel (CACNA1C, CACNA1D, CACNA2D1), potassium channel (KCNH2, KCNQ1), and sodium channel (SCN5A) genes, although the large number of affected genes is consistent with broad spliceosomal perturbation and a formal enrichment test would be needed to determine whether cardiac action potential genes are preferentially affected. Integration of RIP-seq data (597 SF3B2-enriched transcripts, FDR < 0.05) with splicing analysis identified CACNA2D1, which encodes an auxiliary subunit of L-type calcium channels, as both directly bound by SF3B2 and alternatively spliced upon knockdown. ConclusionThese findings identify SF3B2 as a regulator of cardiomyocyte calcium handling and suggest that SF3B2-dependent missplicing of CACNA2D1 may link core spliceosome function to the splicing programs underlying cardiomyocyte functional maturation. Significance StatementThis study provides the first systematic functional screen of splicing factors in cardiomyocyte calcium handling and identifies SF3B2, a U2 snRNP subunit, as a regulator of cardiac ion channel splicing. Transcriptomic, splicing, and RNA binding data converge on CACNA2D1, identifying a route by which a core splicing factor shapes cardiac contractility, extending cardiac splicing regulation beyond the accessory RBPs studied to date.

developmental biology↗

bioRxiv2026

Plasmodium blood-stage induces trained immunity in hepatocytes

Plasmodium parasites, the causative agents of malaria, are transmitted at high levels in endemic areas and sequential infections are common. Using a mouse model of infection we discovered liver burden was suppressed in blood-stage experienced, compared to naive, animals, independently of adaptive responses and inflammation. We observed greater chromatin accessibility of a subset of interferon stimulated genes in blood-stage experienced animals, and a rapid increase in transcription of these genes upon sporozoite challenge. Ex vivo stimulation of hepatocytes from blood-stage experienced mice also led to a rapid and elevated response upon treatment with an unrelated antigen, ctDNA. Taken together, these data are consistent with a model in which hepatocytes are reprogramed by blood stage Plasmodium infection to exhibit trained memory akin to what has been described in innate immune cells. The consequences of training of hepatocytes could be wide-reaching and might alter responses to diverse pathogens and other stimuli.

immunology↗

bioRxiv2026

Spatial Decoding of Tertiary Lymphoid Structure Maturation in Non-Small Cell Lung Cancer Using Deep Neural Networks

Understanding the role of tertiary lymphoid structures (TLS) is crucial in non-small cell lung cancer (NSCLC), as they are associated with patient prognosis and treatment outcomes. Specific cellular ecosystems that originate anti-tumor activity or predict immunotherapy response remain poorly characterized. To this end, we developed a high-resolution, multimodal spatial atlas jointly profiling transcriptomics, proteomics, and histology to characterize TLS maturation in NSCLC alongside secondary lymph organs as a baseline. Using this atlas, we proposed a pathologist-in-the-loop framework that combines a variational graph autoencoder (VGAE) with diffusion pseudotime to refine human expert annotations and characterize TLS maturation. These spatial molecular representations were extended to H&E whole-slide images via a vision transformer-based foundation model. Next, we resolved cellular composition, spatial organization, and cell-cell interactions within these data and defined two divergent spatial ecosystems. Clinical evidence suggests that these ecosystems are associated with distinct patient outcomes: a mature germinal center niche with favorable prognoses, and a tumor-macrophage-fibroblast niche with unfavorable prognoses. In summary, our work decodes key components of TLS heterogeneity, identifies hallmark spatial patterns involved in NSCLC adaptive immunity, and provides a framework for translating spatial omics insights into clinical applications.

genomics↗

bioRxiv2026

FKS1/2-variant independent mechanisms underlying the emergence of resistance in echinocandin-refractory Candida auris infections

The emerging fungus Candida auris is a drug resistant global public health threat and WHO critical priority pathogen. Recommended first-line invasive candidiasis treatment is echinocandin monotherapy, but C. auris can develop on-treatment resistance via FKS1/2 gene mutations and additional, previously unexplained mechanisms. To better understand echinocandin failure in C. auris, we sequenced the genomes of echinocandin refractory FKS1/2 wild-type C. auris serial isolates from two critically unwell patients in London, UK. Population analysis profiling revealed echinocandin heteroresistance, and in vitro culture of clinical isolates at supra-MIC concentrations of anidulafungin (8 g/ml) exhibited morphotypic heterogeneity. Small colony variants (SCVs) and large colony variants (LCVs) showed elevated MICs with polyploidy (to 4n and above) alongside adaptive changes in cell wall {beta}-1,3-glucan content. LCVs contained significantly more mutations in calcineurin-related stress tolerance pathway gene CRZ1 compared to clinical parents and SCVs, associated with further increases in MIC. These findings indicate progressive step-wise accrual of adaptation to echinocandins, including genomic instability, alterations in stress tolerance pathways, and cell wall remodeling, paving the way for resistance emergence. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/700071v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14460b7org.highwire.dtl.DTLVardef@29a598org.highwire.dtl.DTLVardef@36d187org.highwire.dtl.DTLVardef@f62c8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Early-life stromal niches orchestrate B lymphopoiesis at the brain's borders

The dura mater serves as a critical immunological niche for the central nervous system, yet the mechanisms governing the emergence of this niche in early life remain understudied. Here, we chart the trajectory of dural immune development, uncovering a distinctive function for the murine dura as a transient niche for B lymphopoiesis in the early-postnatal window. Shared embryonic progenitors initiate dural B cell development in concert with a multi-organ wave of extramedullary lymphopoiesis that contributes distinctively to the peripheral B cell pool. In the dura, B cells develop locally in discrete sinus-proximal foci, occupying an anatomically defined and developmentally restricted fibroblast niche. Sinus-proximal fibroblasts express the pro-hematopoietic chemokine CXCL12, and local deletion of this crucial factor severely impairs dural B lymphopoiesis. These data reveal a critical function for dural fibroblasts in shaping the early-life B cell compartment and provide a model for how extramedullary niches may support early-life leukocyte production.

immunology↗

bioRxiv2025

Synaptic histamine shapes the neurocomputational dynamics of human learning

Histamine was the first canonical monoamine identified in the mammalian brain 1-6, yet arguably remains the least understood in its mechanistic contributions to human behaviour. Using a first-in-class causal probe (H3R inverse agonist pitolisant), we show how increased synaptic histamine shapes offline and online temporal-hippocampal dynamics, sustaining learning-related activity and polarising retrieval computations. At a broader level, elevated histamine adaptively shifts neurocomputational strategy under high cognitive load, while stabilising value updates during aversive reinforcement learning. These findings uncover a mechanistically grounded influence of this underexplored system on human neurocomputation, supporting its therapeutic potential in psychiatry.

neuroscience↗

bioRxiv2025

In vivo bioreactor matures iPSC-CMs for MLP disease modeling

Muscle LIM protein (MLP) is a critical regulator of cardiomyocytes (CMs) cytoarchitecture, and its deficiency results in late-onset dilated cardiomyopathy (DCM) in both mice and human. However, recapitulating this phenotype in vitro using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has been challenging largely due to their immature state. Here, we generated MLP knockout (MLP-KO) mouse iPSCs and differentiated them into CMs. We then employed both conventional in vitro and in vivo transplantation approach using immunocompromised rat hearts to promote cardiomyocytes maturation. Our results showed that while in vitro-matured MLP-KO iPSC-CMs failed to exhibit disease phenotypes, in vivo-matured MLP-KO iPSC-CMs successfully recapitulated the hallmarks of DCM, including disrupted sarcomeric architecture and upregulation of atrial natriuretic peptide (ANP), closely mirroring disease progression observed in MLP-deficient mice. These findings demonstrate that the in vivo maturation environment is essential for the maturation of iPSC-derived cardiomyocytes to better model genetic cardiac diseases like DCM and provide valuable insights for future therapeutic strategies.

bioengineering↗

bioRxiv2025

Single-cell transcriptomics reveals the impact of sex and age in the healthy human liver

Background & AimsThe liver is a vital organ composed of parenchymal, nonparenchymal, and immune cell populations. Single-cell sequencing approaches now provide the opportunity to understand how sex and age influence gene expression and cellular function across cell types within the liver. MethodsWe analyzed the cellular composition and interactions for the human liver through single-nucleus RNA sequencing (snRNA-seq), incorporating insights from 37 healthy liver samples. The dataset contains cells from female and male donors spanning more than seven decades of life, and analysis was performed to evaluate the impact of sex and age on differential gene expression, pathway enrichment, and predicted ligand-receptor and protein-protein interactions. ResultsExcluding the X and Y chromosomes, we identified 374 genes uniquely enriched in cells of the female liver and 520 genes enriched in cells of the male liver. Differential expression analysis defined unique circuitries enriched within each cell type between females and males and their impact on cell-cell communication and response to external signals, including enrichment of cholesterol/lipid metabolism, transforming growth factor beta (TGF-beta) signaling, and fibronectin (FN1) production in female cells and bone morphogenic protein (BMP) signaling in male cells. With increased age, we observe a greater diversity in gene expression, including enrichment of genes that regulate neuregulin (NGR) signaling at older ages, while genes regulating insulin growth factor (IGF) signaling are enriched at younger ages. Senescence signatures were also identified for each cell type within the liver. ConclusionsThese results define the activities of healthy cell types within the liver across sex and age and provide a foundation for studies to examine how ancestry, geography, and disease states influence liver function within these contexts. Impact and ImplicationsOur study analyzes 37 human liver samples at the single-cell level to understand how sex and age influence gene expression, cell interactions, and response to signals across liver cell types and sub-types. These findings are of particular significance for researchers who need to understand how sex and age may influence the response of individual cell types to injury or treatment of injury. This dataset will also provide a healthy reference for future studies to understand how ancestry, geography, and disease states shape liver biology across age and sex.

cell biology↗

bioRxiv2024

Inhibition of SF3B1 affects recruitment of P-TEFb to chromatin through multiple mechanisms

Processing of nascent pre-mRNAs is tightly coupled to transcription by RNA polymerase II (RNAPII) through reversible phosphorylation of the polymerase and associated factors by transcriptional kinases. P-TEFb, comprising cyclin-dependent kinase (CDK)9 and cyclin T1, is a key transcription elongation kinase, which also regulates co-transcriptional splicing and mRNA cleavage and polyadenylation. Chemical inhibition of SF3B1, a component of the splicing factor U2 snRNP, decreases P-TEFb recruitment to chromatin and mirrors the effect of P-TEFb inhibition on transcription. However, the mechanism of this effect of SF3B1 inhibitors was unclear. Here we show that SF3B1 inhibition causes rapid nuclear export of P-TEFb and loss of SF3B1 phosphorylation. SF3B1 is in complex with P-TEFb on chromatin with the elongation/splicing factor HTATSF1 and the splicing factor SNW1. SF3B1 inhibition causes the nuclear export of SNW1, but not of HTATSF1. The chromatin association of AFF4, an interaction partner of P-TEFb, is also affected by SF3B1 inhibition. Surprisingly, SF3B1 inhibition promotes degradation of SRSF2, a splicing factor known to help recruit P-TEFb to chromatin. Our results indicate that SF3B1 inhibition affects P-TEFb recruitment to genes via multiple pathways. Together, these interactions ensure efficient coupling of transcription and splicing.

molecular biology↗

bioRxiv2024

Tolerance thresholds underlie responses to DNA damage during germline development

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we find that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbour unexpectedly few new TE insertions, despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other non-coding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.

developmental biology↗

bioRxiv2023

Acod1 Expression in Cancer Cells Promotes Immune Evasion through the Generation of Inhibitory Peptides

Targeting PD-1 is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment (TME). Here, we find that PD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to PD-1, as decreasing Acod1 levels in PD-1 resistant cancer cells can sensitize tumors to PD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on secretion of ITA, but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

cancer biology↗

bioRxiv2023

Overcome Prostate Cancer Resistance to Immune Checkpoint Therapy with Ketogenic Diet-Induced Epigenetic Reprogramming

Advanced prostate cancer (PCa) is overwhelmingly resistant to immune checkpoint blockade (ICB) therapy, representing a formidable clinical challenge. In this study, we developed a syngeneic murine PCa model with acquired ICB resistance. Using this model, synergistic efficacy was achieved by combining anti-PD1 and anti-CTLA4 antibodies with histone deacetylase inhibitor (HDACi) vorinostat, a cyclic ketogenic diet (CKD), or supplementation of ketone body {beta}-hydroxybutyrate (BHB, endogenous HDACi) via 1,3-butanediol-admixed food. CKD and BHB supplementation delayed PCa tumors as monotherapy, and both BHB and adaptive immunity are required for the anti-tumor activity of CKD. Single-cell transcriptomic and proteomic profiling revealed that the HDACi and ketogenesis-enhanced ICB therapy involves cancer-cell-intrinsic (upregulated MHC class I molecules) and extrinsic mechanisms (CD8+ T cell chemoattraction, M1/M2 macrophage rebalancing, monocyte differentiation toward antigen presenting cells, and diminished neutrophils). Overall, these findings underscore the potential of using HDACi and optimized KD to enhance ICB therapy for PCa.

cancer biology↗

bioRxiv2023

High-resolution molecular atlas of a lung tumor in 3D

Cells live and interact in three-dimensional (3D) cellular neighborhoods. However, histology and spatial omics methods mostly focus on 2D tissue sections. Here we present a 3D spatial atlas of a routine clinical sample, an aggressive human lung carcinoma, by combining in situ quantification of 960 cancer-related genes across [~]340,000 cells with measurements of tissue-mechanical components. 3D cellular neighborhoods subdivided the tumor microenvironment into tumor, stromal, and immune multicellular niches. Interestingly, pseudotime analysis suggested that pro-invasive epithelial-to-mesenchymal transition (EMT), detected in stroma-infiltrating tumor cells, already occurred in one region at the tumor surface. There, myofibroblasts and macrophages specifically co-localized with pre-invasive tumor cells and their multicellular molecular signature identified patients with shorter survival. Moreover, cytotoxic T-cells did not infiltrate this niche but colocalized with inhibitory dendritic and regulatory T cells. Importantly, systematic scoring of cell-cell interactions in 3D neighborhoods highlighted niche-specific signaling networks accompanying tumor invasion and immune escape. Compared to 2D, 3D neighborhoods improved the characterization of immune niches by identifying dendritic niches, capturing the 3D extension of T-cell niches and boosting the quantification of niche-specific cell-cell interactions, including druggable immune checkpoints. We believe that 3D communication analyses can improve the design of clinical studies investigating personalized, combination immuno-oncology therapies.

systems biology↗

bioRxiv2023

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3UTR (3UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, in particular in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells microenvironment affect 3UTR-APA and IPA in primary human macrophages remains unknown. Here, primary human monocytes were isolated from healthy donors, differentiated and polarized into a pro-inflammatory state and ChrRNA-Seq and 3RNA-Seq were performed to quantify gene expression and characterize new 3UTR-APA and IPA mRNA isoforms. Our results show that polarization of human macrophages from naive to a pro-inflammatory state causes a marked increase both in proximal polyA site selection in the 3UTR and in IPA events, in genes relevant for macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3UTR-APA and IPA mRNA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induce 3UTR-APA alterations. Notably, some of these gene expression differences were also found in tumour-associated macrophages of CRC patients, indicating that they are physiological relevant. Upon macrophage pro-inflammatory polarization SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Our results reveal new 3UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools.

molecular biology↗