Biology subjects

Wei, J.

Publications and source records attributed to Wei, J..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

A chromosome-level genome assembly of Dracaena cambodiana and comparative genomics analysis highlights the distinct biological features of two resource species of dragon's blood

This study reports a chromosome-level genome for Dracaena cambodiana, one of two typical dracaena species in China. This work will help to deepen the understanding of the dracaena species and the conservation and development of plant resources. The final assembly consisted of 54 scaffolds, spanning 1.08 Gb, with a scaffold N50 size of 52.29 Mb, encoded 36860 predicted protein-coding genes. A total of 1,064,810,157 bp of genome sequences were located on 20 chromosomes (2n = 40), accounting for 98.46%. We investigate the possible mechanisms of the longevity of dracaena, the longest-lived plant in the world, which involve DNA repair and post-translational modification. We also performed comparative genomic analysis of the previously assembled genome of Dracaena cochinchinesis with this genome, and found that the two involved interaction of plant-pathogen interaction and ubiquitin mediated proteolysis, which may reveal why Dracaena has environmental adaptability and longevity characteristics.

genomics↗

bioRxiv2024

Microsporidian Nosema bombycis secretes serine protease inhibitor to suppress host cell apoptosis via caspase BmICE

Microsporidia are a group of intracellular pathogens that actively manipulate host cell biological processes to facilitate their intracellular niche. Apoptosis is an important defense mechanism by which host cell control intracellular pathogens. Microsporidia modulating host cell apoptosis has been reported previously, however the molecular mechanism is not yet clear. In this report, we describe that the microsporidia Nosema bombycis inhibits apoptosis of Bombyx mori cells through a secreted protein NbSPN14, which is a serine protease inhibitor (Serpin). An immunofluorescent assay demonstrated that upon infection with N. bombycis, NbSPN14 was initially found in the B. mori cell cytoplasm and then became enriched in the host cell nucleus. Overexpression and RNA-interference (RNAi) of NbSPN14 in B. mori embryo cells confirmed that NbSPN14 inhibited host cell apoptosis. Immunofluorescent and Co-IP assays verified the co-localization and interaction of NbSPN14 with the BmICE, the caspase 3 homolog in B. mori. Knocking out of BmICE or mutating the BmICE-interacting P1 site of NbSPN14, eliminated the localization of NbSPN14 into the host nucleus and prevented the apoptosis-inhibiting effect of NbSPN14, which also proved that the interaction between BmICE and NbSPN14 occurred in host cytoplasm and the NbSPN14 translocation into host cell nucleus is dependent on BmICE. These data elucidate that N. bombycis secretory protein NbSPN14 inhibits host cell apoptosis by directly inhibiting the caspase protease BmICE, which provides an important insight for understanding pathogen-host interactions and a potential therapeutic target for N. bombycis proliferation. Author SummaryMicrosporidia constitute a class of eukaryotic pathogens that exclusively reside within host cells. The species Nosema bombycis is the first microsporidian identified as the pathogen of silkworm Pebrine disease. In our research, we discovered how N. bombycis cleverly evades the hosts defenses. It has developed a strategy to survive inside host cells by manipulating host cell apoptosis, disarming the host cells self-destruct mechanism. In this study, we discovered that the N. bombycis secretes a serine protease inhibitor named NbSPN14, which infiltrates the cytoplasm of the host cell. The NbSPN14 interacts with the executioner Caspase protease BmICE within the silkworms apoptotic pathway, effectively neutralizing its apoptoic activity and thus curbing the apoptosis of the host cells.

microbiology↗

bioRxiv2024

RTM-align: an improved RNA alignment tool with enhanced short sequence performance via post-standardization and fragment alignment

Understanding the three-dimensional structure of RNA is crucial for studying various biological processes. Accurate alignment and comparison of RNA structures are essential for illustrating RNA functionality and evolution. The existing RNA alignment tools suffer from limitations such as size-dependency of scoring functions and inadequate handling of short RNA fragments, leading to the conflicting interpretation of structural and functional relationships among RNA molecules. Hence, we introduce RTM-align, a novel RNA structural alignment tool enhanced for short RNAs. RTM-align employs the RTM-score, which integrates post-standardization to ensure size-independence and utilizes a fragment alignment strategy that improves alignment accuracy by concentrating on structural motifs and local structural elements. Benchmarking results demonstrate that RTM-align outperforms existing tools in RNA structure comparison, offering a universal scoring scale regardless of RNA length. The improvement is particularly evident in evaluating predicted structures for CASP15 RNA targets, with significant enhancements observed for the short RNA target R1117. RTM-align is expected to significantly improve the accuracy and reliability of RNA structure alignment and comparison, thereby aiding in the deeper understanding and discovery of novel RNA functions and their interactions in biological systems. RTM-align is now available at https://github.com/BEAM-Labs/RTM-align.

bioinformatics↗

bioRxiv2024

Activity of prefrontal cortex serotonin 2A receptor expressing neurons is necessary for the head-twitch response of mice to psychedelic drug DOI in a sex-dependent manner

Serotonin 2A receptors (5-HT2ARs) mediate the effects of psychedelic drugs. 5-HT2AR agonists, such as (-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI), that produce a psychedelic experience in humans induce a head-twitch response (HTR) behavior in rodents. However, it is unknown whether the activity of 5-HT2AR expressing neurons is sufficient to produce the HTR in the absence of an agonist, or in which brain region 5-HT2ARs control the HTR. Here, we use an optogenetic approach to examine whether activation of 5-HT2AR expressing neurons in the mouse prefrontal cortex (PFC) is sufficient to induce HTRs alone, or may augment the HTR produced by DOI, and if inhibition of these neurons prevents DOI-induced HTRs in mice. We crossed Htr2a-Cre mice to Cre-dependent optogenetic lines Ai32 (channelrhodopsin) and Ai39 (halorhodopsin) to selectively activate and inhibit (respectively) 5-HT2AR-expressing neurons in the PFC of adult mice. We found that optogenetic stimulation of PFC 5-HT2AR expressing neurons in the absence of an agonist does not increase HTRs in mice. In both male and female Ai32 mice that received vehicle, there was no difference in HTRs in mice that expressed Htr2a-Cre compared with control mice, indicating that optogenetic activation of 5-HT2AR+ cells in the PFC was not sufficient to produce HTRs in the absence of an agonist. In female mice, activation of PFC 5-HT2AR expressing neurons augmented the HTR produced by DOI. However, this result was not seen in male mice. In contrast, inhibition of 5-HT2AR expressing neurons in the PFC prevented the increase in HTR produced by DOI in male, but not in female, mice. Together, these findings suggest that activation of 5-HT2ARs in the PFC is not sufficient to induce HTRs in the absence of a 5-HT2AR agonist but is necessary for induction of HTRs by a 5-HT2AR agonist in a sex-dependent manner.

neuroscience↗

bioRxiv2024

Axo-axonic synaptic input drives homeostatic plasticity by tuning the axon initial segment structurally and functionally

The stability of functional brain network is maintained by homeostatic plasticity, which restores equilibrium following perturbation. As the initiation site of action potentials, the axon initial segment (AIS) of glutamatergic projection neurons (PyNs) undergoes dynamic adjustment that exerts powerful control over neuronal firing properties in response to changes in network states. Although AIS plasticity has been reported to be coupled with the changes of network activity, it is poorly understood whether it involves direct synaptic input to the AIS. Here we show that changes of GABAergic synaptic input to the AIS of cortical PyNs, specifically from chandelier cells (ChCs), are sufficient to drive homeostatic tuning of the AIS within 1-2 weeks, while those from parvalbumin-positive basket cells do not. This tuning is reflected in the morphology of the AIS, the expression level of voltage-gated sodium channels, and the intrinsic neuronal excitability of PyNs. Interestingly, the timing of AIS tuning in PyNs of the prefrontal cortex corresponds to the recovery of changes in social behavior caused by alterations of ChC synaptic transmission. Thus, homeostatic plasticity of the AIS at postsynaptic PyNs may counteract deficits elicited by imbalanced ChC presynaptic input. TeaserAxon initial segment dynamically responds to changes in local input from chandelier cells to prevent abnormal neuronal functions.

neuroscience↗

bioRxiv2024

PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinsons disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14-3-3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Strikingly, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

neuroscience↗

bioRxiv2024

Circular RNA aptamers ameliorate AD-relevant phenotypes by targeting PKR

Here, we delineated the remarkably elevated neuroinflammation accompanied by progressive activation of double-stranded RNA (dsRNA)-activated Protein Kinase R (PKR) and PKR-related dsRNA pathways in hippocampus of 5xFAD mice upon Alzheimers disease (AD) progression. AAV-delivery of circular RNAs possessing short-imperfect duplex regions (ds-cRNAs) to neurons and microglia effectively dampened excessive PKR activity with little toxicity, accompanying with reduced neuroinflammation and amyloid-beta (A{beta}) plaques, resulting in neuroprotection and enhanced capability of spatial learning and memory in AD mouse models. These findings suggest a therapeutic potential of ds-cRNA aptamers as PKR inhibitors in AD therapy.

molecular biology↗

bioRxiv2024

Multivalent 28S rRNA Is the Organizer of the Nucleolus's Multi-layered Architecture

The nucleolus widely exists in all eukaryotic species. Throughout evolution, two types of nucleoli have emerged: bipartite, which has two-layered subcompartments, and tripartite, which features three nested sub-compartments: granular component (GC), dense fibrillar component (DFC), and fibrillar center (FC). FC and DFC form a core-shell architecture and are immersed by a large GC. However, factors that mediated the formation of the three-layered architecture remain largely unknown. Here, we discovered that rRNAs have specific localizations within the nucleolus, with 28S rRNA forming assemblies surrounding the DFC. Multivalent interactions of 28S rRNA drove the formation of the hollow shell structure of DFC, which mediated the development of a three-layered structure resembling nucleolar GC-DFC-FC. Moreover, in organisms with a tripartite nucleolus, 28S rRNA has evolved to acquire a higher degree of multivalency. Our data implies that the emergence of multivalency of 28S rRNA facilitates the transition of bipartite to tripartite nucleolus.

cell biology↗

bioRxiv2024

Physics-driven structural docking and protein language models accelerate antibody screening and design for broad-spectrum antiviral therapy

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline AbGen that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. Our AbGen centers around a novel antibody language model (AbLM) that is pretrained on 12 million generic protein domain sequences and fine-tuned on 4,000+ paired VH-VL sequences, with IgG-specific CDR-masking and VH-VL cross-attention. AbLM provides a latent space of IgG sequence embeddings for AbGen, including (a) landscapes of IgGs activities in neutralizing the wild-type virus are analyzed through structure prediction for IgG and IgG-antigen (viral protein spikes receptor binding domain, RBD) interactions; and (b) landscapes of IgGs susceptibility in neutralizing variant viruses are predicted through Gaussian process regression, despite that as few as 14 clinical antibodies responses to variants of concern are available. The AbGen pipeline was applied to over 1300 IgG sequences we collected from RBD-binding B cells of convalescent patients. With experimental validations, AbGen efficiently prioritized IgG candidates against a broad spectrum of viral variants (wildtype, Delta, and Omicron), preventing the infection of host cells in vitro and hACE2 transgenic mice in vivo. Compared to other existing protein language models that require 10-100 times more model parameters, AbLM improved the precision from around 50% to 75% to predict IgGs with low variant susceptibility. Furthermore, AbGen enables structure-based computational protein redesign for selected IgG clones with single amino acid substitutions at the RBD-binding interface that doubled the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screen and re- design combining data-driven protein language models and Kriging for antibody sequence analysis and activity prediction, in synergy with physics-driven protein docking and design for antibody-antigen interface analyses and functional optimization.

immunology↗

bioRxiv2024

Targeting GFPT2 to reinvigorate immunotherapy in EGFR-mutated NSCLC

In the evolving field of cancer immunotherapy, EGFR-mutated NSCLC presents a significant challenge, demonstrating marked innate resistance to established treatments. An effective method to counter this resistance remains elusive. Through comprehensive genetic and pharmacological analyses across various models, we have identified glutamine fructose-6-phosphate transaminase 2 (GFPT2) as a key facilitator of immune evasion in EGFR-mutated NSCLC. Mechanistically, under EGFR mutation condition, GFPT2 expression, which is typically low in normal tissues, is highly induced via EGFR/IRE1/Xbp1s signaling axis, leading to a significant increase in intracellular UDP-GlcNAc and consequently, an altered N-glycosylation profile. GFPT2 escalates the expression and glycosylation of PD-L1, PVR and CD276, bolstering their interactions with CD8+T cells, and amplifies CD73 glycosylation, thereby intensifying adenosine-mediated CD8+T cells suppression. These actions collectively reduce tumor cell vulnerability to CD8+T cell-mediated death. Moreover, GFPT2 regulates EGFR glycosylation, which consequentially modulates the EGFR-dependent secretion of CXCL10 and VEGF, thus impeding CD8+T cell infiltration within tumors. We further identified a GFPT2 isoform-specific inhibitor that potentiates PD-1 blockade therapy beyond that of existing strategy, corroborated by results in xenografts and patient-derived organoids. Together, these findings illuminate the promising therapeutic potential of GFPT2 as a metabolic checkpoint, offering an innovative approach to invigorate immunotherapy in NSCLC with EGFR mutations.

cancer biology↗

bioRxiv2024

Alterations in genes associated with cytosolic RNA sensing in whole blood are associated with coronary microvascular disease in SLE

ObjectiveTo investigate whether gene signatures discriminate systemic lupus erythematosus (SLE) patients with coronary microvascular dysfunction (CMD) from those without and whether any signaling pathway is linked to the underlying pathobiology of SLE CMD. MethodsThis study collected whole blood RNA samples from female subjects aged 37 to 57, comprising 11 SLE patients (4 SLE-CMD, 7 SLE-non-CMD) and 10 HC. Total RNA was then used for library preparation and sequencing. Differential gene expression analysis was performed to identify gene signatures associated with CMD in SLE patients using DEseq2 v1.42.0. Gene Set Enrichment Analysis were performed by ClusterProfiler v4.10.0 and pathfindR v2.3.1. ResultsRNA-seq analysis revealed 143 differentially expressed (DE) genes between the SLE and HC groups. GO analysis indicated associations with virus defense and interferon signaling in SLE. 14 DE genes were identified from comparison between SLE-CMD and SLE-non-CMD with adjusted parameters (padj < 0.1). Notably, SLE-CMD exhibited elevated levels of genes associated with RNA sensing, while downregulated genes in SLE-non-CMD were associated with blood coagulation and cell-cell junction. Further investigation highlighted differences in IFN signaling and ADP-ribosylation pathways between SLE-CMD and SLE-non-CMD, suggesting distinct molecular mechanisms underlying vascular changes in CMD and reduced left ventricular function in non-CMD. ConclusionOur study identified a unique gene signature in SLE-CMD compared to the HC group, highlighting the significant involvement of type 1 interferon, RIG-I family proteins, and chronic inflammation in the progression of SLE-CMD. The intricate relationship between SLE-CMD and these factors underscores their probable role in initiating and advancing SLE-CMD.

bioinformatics↗

bioRxiv2024

TSC22D, WNK and NRBP gene families exhibit functional buffering and evolved with Metazoa for macromolecular crowd sensing

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. Myriad redundancies have evolved across all facets of osmosensing in metazoans, including among water and ion transporters, regulators of cellular morphology, and macromolecular crowding sensors, hampering efforts to gain a clear understanding of how cells respond to rapid water loss. In this study, we harness the power of gene co-essentiality analysis and genome-scale CRISPR-Cas9 screening to identify an unappreciated relationship between TSC22D2, WNK1 and NRBP1 in regulating cell volume homeostasis. Each of these genes have paralogs and are functionally buffered for macromolecular crowd sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK and NRBP family members physically associate into cytoplasmic biocondensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans reveals that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is concomitant with rapid IDR length expansion in WNK family kinases. Our study identifies functions for unrecognized components of the cell volume sensing machinery and reveals that TSC22D, WNK and NRBP genes evolved as cytoplasmic crowding sensors in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

cell biology↗

bioRxiv2024

Systematic Analysis of Immune Changes after B-cell Depletion in Autoimmune Multiple Sclerosis

Multiple sclerosis (MS) is a complex genetically mediated autoimmune disease of the central nervous system where anti-CD20-mediated B cell depletion is remarkably effective in the treatment of early disease. While previous studies investigated the effect of B cell depletion on select immune cell subsets using flow cytometry-based methods, the therapeutic impact on patient immune landscape is unknown. In this study, we explored how a therapy-driven "in vivo perturbation" modulates the diverse immune landscape by measuring transcriptomic granularity with single-cell RNA sequencing (scRNAseq). We demonstrate that B cell depletion leads to cell type-specific changes in the abundance and function of CSF macrophages and peripheral blood monocytes. Specifically, a CSF-specific macrophage population with an anti-inflammatory transcriptomic signature and peripheral CD16+ monocytes increased in frequency post-B cell depletion. This was accompanied by increases in TNF messenger RNA and protein in monocytes post-B cell depletion, consistent with the finding that anti-TNF treatment exacerbates autoimmune activity in MS. In parallel, B cell depletion induced changes in peripheral CD4+ T cell populations, including increases in the frequency of TIGIT+ regulatory T cells and marked decreases in the frequency of myelin peptide loaded-tetramer binding CD4+ T cells. Collectively, this study provides an exhaustive transcriptomic map of immunological changes, revealing different mechanisms of action contributing to the high efficacy in B cell depletion treatment of MS.

immunology↗

bioRxiv2024

IL-3-driven T cell-basophil crosstalk enhances anti-tumor immunity

Cytotoxic T lymphocytes (CTLs) are pivotal in combating cancer, yet their efficacy is often hindered by the immunosuppressive tumor microenvironment, resulting in exhaustion. This study investigates the role of interleukin (IL)-3 in orchestrating anti-tumor immunity through CTL modulation. Intratumoral CTLs undergo a progressive decline in IL-3 production, which is correlated with impaired cytotoxic function. Augmenting IL-3, through intraperitoneal administration, IL-3-expressing melanoma cells, or IL-3-engineered CD8+ T cells, confers protection against tumor progression, concomitant with increased CTL activity. CTLs are critical in this therapeutic efficacy as IL-3 demonstrates no impact on tumor growth in RAG1 knockout mice or following CD8+ T cell-depletion. Rather than acting directly, CTL-derived IL-3 exerts its influence on basophils, synergistically amplifying anti-tumor immunity within CTLs. Introducing IL-3-activated basophils retards tumor progression, whereas basophil depletion diminishes the effectiveness of IL-3 supplementation. Furthermore, IL-3 prompts basophils to produce IL-4, which subsequently elevates IFN-{gamma} production and viability of CTLs. Notably, the importance of basophil-derived IL-4 is evident from the absent benefits in IL-3-supplementated, IL-4 knockout tumor-bearing mice. Overall, this research unveils IL-3-mediated CTL-basophil crosstalk in regulating anti-tumor immunity and offers the prospect of harnessing IL-3 sustenance as a promising approach for optimizing and enhancing cancer immunotherapy. Significance StatementThis study elucidates the critical role of IL-3 in orchestrating anti-tumor immunity, particularly within the context of CTLs and melanoma growth. It reveals a progressive decline in CTL-derived IL-3 during tumor progression, correlated with CTL exhaustion--a formidable barrier in cancer immunotherapy. Intriguingly, augmentation of IL-3, achieved through diverse means, effectively impedes tumor progression by enhancing CTL activity. This research unveils a novel mechanism: IL-3-mediated crosstalk between CTLs and IL-4-producing basophils, resulting in the rejuvenation of CTLs and amplifying their anti-tumor ability. These insights hold promise for the advancement and optimization of cancer immunotherapeutic strategies, deepening our comprehension of CTL dynamics within the tumor microenvironment, and advancing our ability to combat cancer effectively.

immunology↗

bioRxiv2024

The Alzheimer's Aβ peptide forms biomolecular condensates that trigger amyloid aggregation

The onset and development of Alzheimers disease (AD) is linked to the accumulation of pathological aggregates formed from the normally monomeric amyloid-{beta} peptide within the central nervous system. These A{beta} aggregates are increasingly successfully targeted with clinical therapies, but the fundamental molecular steps that trigger the initial nucleation event leading to the conversion of monomeric A{beta} peptide into pathological aggregates remain unknown. Here we show that the A{beta} peptide can form biomolecular condensates on lipid bilayers both in molecular assays and in living cells. Our results reveal that these A{beta} condensates can significantly accelerate the primary nucleation step in the amyloid conversion cascade that leads to the formation of amyloid aggregates and plaque. We show that A{beta} condensates contain phospholipids, are intrinsically heterogenous, and are prone to undergo a liquid-to-solid transition leading to the formation amyloid fibrils. These findings uncover the liquid-liquid phase separation behaviour of the A{beta} peptide, and reveal a new molecular step very early in the amyloid-{beta} aggregation cascade that can form the basis for novel therapeutic intervention strategies. Significance statementThe hallmark of Alzheimers disease is the abnormal buildup of the normally soluble amyloid {beta} protein aggregates in the central nervous system. While the molecular mechanisms at the late stages of the amyloid {beta} aggregation cascade are well understood, the initial steps remained elusive until now. Our current study demonstrates that amyloid {beta} undergoes liquid-liquid phase separation on lipid surfaces, which triggers primary nucleation and initiates the amyloid {beta} aggregation cascade. This newly identified step in the molecular mechanism of Alzheimers disease represents a promising target for the development of alternative innovative therapeutic strategies.

biophysics↗

bioRxiv2024

GTPase-activating protein ARAP1 regulates circular dorsal ruffles as a nutrient uptake mechanism in the Hep3B hepatocellular carcinoma cell line

Circular dorsal ruffles (CDRs), large-scale rounded membrane ruffles, function as precursors of macropinocytosis. We recently reported that CDRs are exposed in the Hep3B hepatocellular carcinoma cell line, while not in other hepatocellular carcinoma cell lines, indicating that the CDRs in Hep3B are associated with malignant potential. In this study, we investigated the cellular function of CDRs in Hep3B cells by focusing on the molecular mechanisms of the GTPase-activating protein ARAP1. ARAP1 was localized to the CDRs, the sizes of which were reduced by deletion of this protein. High-resolution scanning electron micrographs revealed that CDRs comprise small vertical lamellipodia, the expression pattern of which was disrupted in ARAP1 KO cells. Extracellular solute uptake, rate of cell growth, and malignant potential were attenuated in the KO cells. ARAP1 is also localized in Hep3B cell mitochondria, although not in those of the Huh7 hepatocellular carcinoma cell line. On the basis of these findings, we propose that the aberrant expression of ARAP1 in Hep3B cells modulates CDRs, thereby resulting in an excess uptake of nutrients as an initial event in cancer development. SUMMARY STATEMENTARAP1 regulates circular dorsal ruffles (CDRs) in the Hep3B HCC cell line and deletion of this protein attenuates malignant potential, thereby indicating the involvement of CDRs in cancer development.

cell biology↗

bioRxiv2023

Functional characterization of eQTLs and asthma risk loci with scATAC-seq across immune cell types and contexts

Cis-regulatory elements (CREs) control gene transcription dynamics across cell types and in response to the environment. In asthma, multiple immune cell types play an important role in the inflammatory process. Genetic variants in CREs can also affect gene expression response dynamics and contribute to asthma risk. However, the regulatory mechanisms underlying control of transcriptional dynamics across different environmental contexts and cell-types at single cell resolution remains to be elucidated. To resolve this question, we performed scATAC-seq in activated peripheral blood mononuclear cells (PBMC) from 16 children with asthma with phytohemagglutinin (PHA) or lipopolysaccharide (LPS), and treated with dexamethasone (DEX), an antiinflammatory glucocorticoid. We analyzed changes in chromatin accessibility, measured transcription factor motif activity, and identified treatment and cell-type specific transcription factors that drive changes in both gene expression mean and variability. We observed strong positive linear dependence between motif response and their target gene expression changes, but negative in variability changes. This result suggests that an increase of transcription factor binding tightens the variability of gene expression around the mean. We then annotated genetic variants in chromatin accessibility peaks and response motifs followed by computational fine-mapping of eQTL signals from a pediatric asthma cohort. We found that eQTLs were 5-fold enriched in peaks with response motifs and refined the credible set for 410 asthma risk genes, with 191 having the causal variant in response motifs. In conclusion, scATAC-seq enhances the understanding of molecular mechanisms for asthma risk variants mediated by gene expression.

genomics↗

bioRxiv2023

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing (eCLIP-seq) to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than one hundred genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with regulatory proteins (RsmA, AlgU, NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCENumerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing (eCLIP-seq) was employed to identify the Hfq-binding sites and potential targets in the root-associated P. stutzeri A1501, and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown that Hfq interactions with various regulatory proteins and their potential targets at both the protein and RNA levels. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

microbiology↗