Biology subjects

Fang, J.

Publications and source records attributed to Fang, J..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts

BACKGROUNDDiabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor (TNF) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and {beta}-oxidation. Here we examined how HDAC6 regulates TNF production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODSHDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNF and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTSMyocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNF levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNF with an anti-TNF monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNF levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNF levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONSAugmenting HDAC6 activity inhibits mCI activity by increasing TNF levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIschemic heart disease (IHS) is a leading cause of death globally, and its presence in diabetic patients is a grievous combination, leading to high mortality and heart failure. C_LIO_LIDiabetes impairs assembly of mitochondrial complex I (mCI), complex III dimer, and complex IV monomer into the respiratory chain supercomplexes, resulting in electron leak and the formation of reactive oxygen species (ROS). C_LIO_LIBy oxidizing reduced nicotinamide adenine dinucleotide (NADH) and reducing ubiquinone, mCI physiologically regenerates NAD+ to sustain the tricarboxylic acid cycle and {beta}-oxidation. C_LI What New Information Does This Article Contribute?O_LIMyocardial ischemia/reperfusion injury (MIRI) and diabetes as comorbidities augment myocardial HDCA6 activity and generation of tumor necrosis factor (TNF), which inhibit myocardial mCI activity. C_LIO_LIGenetic disruption of histone deacetylase 6 (HDAC6) decreases mitochondrial NADH levels and augments mCI activity in type 1 diabetic mice undergoing MIRI via decreasing TNF production, leading to decreases in MIRI. C_LIO_LIPretreatment of type 2 diabetic db/db mice with a HDAC6 inhibitor, tubastatin A (TSA), decreases mitochondrial NADH levels and augments mCI activity by decreasing TNF levels, leading to improvements in cardiac function. C_LI Patients with diabetes are more susceptible to MIRI than non-diabetics with greater mortality and resultant heart failure. There is an unmet medical need in diabetic patients for the treatment of IHS. Our biochemical studies find that MIRI and diabetes synergistically augment myocardial HDAC6 activity and generation of TNF, along with cardiac mitochondrial fission and low bioactivity of mCI. Intriguingly, genetic disruption of HDAC6 decreases the MIRI-induced increases in TNF levels, concomitant with augmented mCI activity, decreased myocardial infarct size, and ameliorated cardiac dysfunction in T1D mice. Importantly, treatment of obese T2D db/db mice with TSA reduces the generation of TNF and mitochondrial fission and enhances mCI activity during reperfusion after ischemia. Our isolated heart studies revealed that genetic disruption or pharmacological inhibition of HDAC6 reduces mitochondrial NADH release during ischemia and ameliorates dysfunction of diabetic hearts undergoing MIRI. Furthermore, HDAC6 knockdown in cardiomyocytes blocks high glucose- and exogenous TNF-induced suppression of mCI activity in vitro, implying that HDAC6 knockdown can preserve mCI activity in high glucose and hypoxia/reoxygenation. These results demonstrate that HDAC6 is an important mediator in MIRI and cardiac function in diabetes. Selective inhibition of HDAC6 has high therapeutic potential for acute IHS in diabetes.

pharmacology and toxicology↗

bioRxiv2023

Chitosan oligosaccharide induces plant resistance gene expression in Pinus massoniana

Chitosan oligosaccharides are the main degradation products from chitosan or chitin and have been reported to induce resistance to diseases in herbaceous plants like cucumber and Arabidopsis. Concomitantly, pine wilt disease is a devastating disease of conifer tree species. Here, we hypothesized that chitosan oligosaccharides induce plant resistance gene (PRG) expression in the woody plant Masson pine, Pinus massoniana. Chitosan oligosaccharides were inoculated into P. massoniana seedlings and the BGISEQ-500 platform was used to generate transcriptomes from chitosan oligosaccharide-treated P. massoniana and control seedlings. A total of 501 differentially expressed genes (DEGs) were identified by comparing the treatment and control groups. A total of 251 (50.1%) DEGs were up-regulated in the treatment relative to the control seedlings and 250 (49.9%) were down-regulated. Inoculation of chitosan oligosaccharide induced the expression of 31 PRGs in P. massoniana seedlings and the relative expression levels of six of the PRGs were verified by RT-qPCR. This is the first study to demonstrate that chitosan oligosaccharide induces the expression of PRGs in a tree species. These results provide important insights into the function of chitosan oligosaccharides and further the prospects of developing a chitosan oligosaccharide-based immune inducer for controlling pine wilt disease.

genomics↗

bioRxiv2022

Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories

Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.

microbiology↗

bioRxiv2022

Monkeypox virus quadrivalent mRNA vaccine induces antibody responses and cellular immunity and protects mice against Vaccinia virus

There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we have developed two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two IMVs (A29L and M1R) and two EEVs (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice have induced MPXV-specific IgG antibodies and potent Vaccinia virus (VACV)-specific neutralizing antibodies. Additionally, it elicited durable MPXV-specific killer memory T-cell immunity as well as memory B-cell immunity in mice. Furthermore, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protected nude mice against the VACV challenge. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP were also protective against the VACV challenge in mice. Overall, our results demonstrated that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.

immunology↗

bioRxiv2022

Single-cell Landscape Analysis of the Circulating Human B Cell Pool under Selective Pressure of Allogeneic Stem Cell Transplantation

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT) chronic graft-versus-host disease (cGVHD), a B cell-mediated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B Cell Receptor (BCR)-activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells under selective pressure of alloantigens, we performed scRNA-Seq analysis on high numbers of purified B cells from allo-HCT patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation and memory. We found striking transcriptional differences in the memory B cell compartment after allo-HCT compared to healthy or infected individuals. To identify intrinsic properties when B-cell tolerance is lost after allo-HCT, we then assessed clusters for differentially expressed genes (DEGs) between patients with vs. without autoimmune-like manifestations (Active cGVHD vs. No cGVHD, respectively). DEGs were found in Active cGVHD in both naive and BCR-activated clusters, suggesting functional diversity. Some DEGs were also differentially expressed across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides new understanding of B-cell memory in the face of chronic alloantigen stimulation. One Sentence SummaryOur scRNA-Seq study of purified B cells after allo-HCT clarifies molecular differences in human B cell subsets when immune tolerance is lost or maintained. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/512162v4_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@5c452eorg.highwire.dtl.DTLVardef@1c1d34borg.highwire.dtl.DTLVardef@171310org.highwire.dtl.DTLVardef@e4bf15_HPS_FORMAT_FIGEXP M_FIG C_FIG The circulating human B cell compartment under the selective pressure of allogeneic hematopoietic stem cell transplantation (Allo-HCT) has an intrinsically altered memory B cell pool. In allo-HCT, genetically disparate donor stem and progenitor cells engraft, regenerating a new peripheral B cell compartment in the host. During ongoing B lymphopoiesis and diversification, selective pressure from alloantigens and other extrinsic factors, including B Cell Activating Factor (BAFF), result in either B cell maturation and immune tolerance (No Chronic GVHD) or altered B cell homeostasis and autoimmune manifestations (Active Chronic GVHD). B cells within defined subsets in patients with Active GVHD have distinct intrinsic programs delineated by differentially expressed genes (DEGs). Some DEGs occur across nearly all B cell subsets ( Differentially Expressed Broadly), while other DEGs are more restricted within only one or a few B cell subsets ( Naive, BCR-activated, or Memory). We affirm altered trajectories for diversification (blue arrow) and enrichment of an atypical memory B cell (ABC) pool, with intrinsic differences when chronic GVHD occurs.

immunology↗

bioRxiv2022

Whole-genome sequencing and single acute toxicity of heavy metal to Photobacterium kishitanii FJ21

In this study, the growth morphology of FJ21 strain was observed, and its 16S rRNA and whole genome were sequenced. Then, related software was used to make genome assembly, gene structure and function annotation, genome phylogenetic tree analysis, genome collinearity analysis and prediction of secondary metabolic gene cluster analysis. Finally, the single acute toxicity of five heavy metals to FJ21 strain was detected. There were luxC, luxD, luxA, luxB, luxF, luxE and luxG genes in FJ21, and the protein encoded by lux operon had certain hydrophilicity. The genome of this strain FJ21 contains a chromosome with a total length of 4853277bp and a GC content of 39.23%. The genome of FJ21 was compared with that of Photobacterium kishitanii ATCCBAA-1194, Photobacterium phosphoreum JCM21184, Photobacterium aquimaris LC2-065, Photobacterium malacitanum CECT9190, and Photobacterium carnosum TMW 2.2021. The average nucleotide identity(ANI), tetra nucleotide signatures (Tetra), comparative genome, and phylogenetic analysis proposed that FJ21 is a strain of Photobacterium kishitanii. In the acute toxicity test, the toxicity of heavy metals to the strain FJ21 is Pb(NO3)2 > ZnSO4{middle dot}7H2O > CdCl2{middle dot}2.5H2O > CuSO4{middle dot}5H2O > K2Cr2O7.

genomics↗

bioRxiv2022

In silico labeling enables kinetic myelination assay in brightfield

Recent advances with deep neural networks have shown the feasibility of acquiring brightfield images with transmitted light and applying in-silico labeling to predict fluorescent images. We have developed a novel in-silico labeling method based on a generative adversarial network and outperforms the state-of-the-art Unet method in generating realistic fluorescent images and quantitatively recapitulating real staining signals, as demonstrated in a complex co-culture myelination assay. Furthermore, we have performed the assay in live mode with multiple kinetic points, applied in-silico labeling to predict fluorescent images from brightfield and quantified the kinetic phenotypic changes. Thus, the proposed approach provides a potential tool to study the kinetics of cellular phenotypic changes with brightfield imaging.

cell biology↗

bioRxiv2022

Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone

Colony stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and thus an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 stimulating osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Single cell RNA- sequencing indicated specific expression of Csf1 in MALPs, which is further increased during aging. To investigate its role, we constructed Csf1 CKO mice using Adipoq-Cre. These mice showed increased femoral trabecular bone over time, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prx1-Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs are a critical player in controlling bone remodeling and hematopoiesis.

cell biology↗

bioRxiv2022

NcPath: A novel tool for visualization and enrichment analysis of human non-coding RNA and KEGG signaling pathways

Noncoding RNAs play important roles in transcriptional processes and participate in the regulation of various biological functions, in particular miRNAs and lncRNAs. Despite their importance for several biological functions, the existing signaling pathway databases do not include information on miRNA and lncRNA. Here, we redesigned a novel pathway database named NcPath by integrating and visualizing a total of 178,308 human experimentally-validated miRNA-target interactions (MTIs), 36,537 experimentally-verified lncRNA target interactions (LTIs), and 4,879 experimentally-validated human ceRNA networks across 222 KEGG pathways (including 27 sub-categories). To expand the application potential of the redesigned NcPath database, we identified 553,523 reliable lncRNA-PCG interaction pairs by integrating co-expression relations, ceRNA relations, co-TF-binding interactions, co-Histone-modification interactions, cis-regulation relations and lncPro Tool predictions between lncRNAs and protein-coding genes. In addition, to determine the pathways in which miRNA/lncRNA targets are involved, we performed a KEGG enrichment analysis using an hypergeometric test. The NcPath database also provides information on MTIs/LTIs/ceRNA networks, PubMed IDs, gene annotations and the experimental verification method used. In summary, the NcPath database will serve as an important and continually updated platform that provides annotation and visualization of the pathways on which noncoding RNAs (miRNA and lncRNA) are involved, and provide support to multimodal noncoding RNAs enrichment analysis. The NcPath database is freely accessible at http://ncpath.pianlab.cn/.

bioinformatics↗

bioRxiv2022

Proteomic Dynamics of Breast Cancers Identifies Potential Therapeutic Protein Targets

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We quantified the proteomes of 76 human BC cell lines using data independent acquisition (DIA) based proteomics, identifying 6091 proteins. We then established a 24-protein panel distinguishing TNBC from other BC types. Integrating prior multi-omics datasets with the present proteomic results to predict the sensitivity of 90 drugs, we found that proteomics data improved drug sensitivity predictions. The sensitivity of the 90 drugs was mainly associated with cell cytoskeleton, signal transduction and mitochondrial function. We next profiled the proteome changes of nine cell lines (five TNBC cell lines, four non-TNBC cell lines) perturbated by EGFR/AKT/mTOR inhibitors. In the TNBC cell lines, metabolism pathways were dysregulated after EGFR/mTOR inhibitors treatment, while RNA modification and cell cycle pathways were dysregulated after AKT inhibitor treatment. Our study presents a systematic multi-omics and in-depth analysis of the proteome of BC cells. This work aims to aid in prioritization of potential therapeutic targets for TNBC as well as to provide insight into adaptive drug resistance in TNBC.

systems biology↗

bioRxiv2022

High-content cellular screen image analysis benchmark study

Recent development of novel methods based on deep neural networks has transformed how high-content microscopy cellular images are analyzed. Nonetheless, it is still a challenge to identify cellular phenotypic changes caused by chemical or genetic treatments and to elucidate the relationships among treatments in an unsupervised manner, due to the large data volume, high phenotypic complexity and the presence of a priori unknown phenotypes. Here we benchmarked five deep neural network methods and two feature engineering methods on a well-characterized public data set. In contrast to previous benchmarking efforts, the manual annotations were not provided to the methods, but rather used as evaluation criteria afterwards. The seven methods individually performed feature extraction or representation learning from cellular images, and were consistently evaluated for downstream phenotype prediction and clustering tasks. We identified the strengths of individual methods across evaluation metrics, and further examined the biological concepts of features automatically learned by deep neural networks.

bioinformatics↗

bioRxiv2022

Separation of phosphatase and kinase activity within the Bub complex is required for proper mitosis

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogues with Bub1 being a kinase while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can supports chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation resulting in reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

cell biology↗

bioRxiv2022

Clustering Deviation Index (CDI): A robust and accurate unsupervised measure for evaluating scRNA-seq data clustering

Single-cell RNA-sequencing (scRNA-seq) technology allows us to explore cellular heterogeneity in the transcriptome. Because most scRNA-seq data analyses begin with cell clustering, its accuracy considerably impacts the validity of downstream analyses. Although many clustering methods have been developed, few tools are available to evaluate the clustering "goodness-of-fit" to the scRNA-seq data. In this paper, we propose a new Clustering Deviation Index (CDI) that measures the deviation of any clustering label set from the observed single-cell data. We conduct in silico and experimental scRNA-seq studies to show that CDI can select the optimal clustering label set. Particularly, CDI also informs the optimal tuning parameters for any given clustering method and the correct number of cluster components.

bioinformatics↗

bioRxiv2021

CV-containing vesicle budding from chloroplast inner envelope membrane is mediated by clathrin but inhibited by GAPC

Clathrin-mediated vesicular formation and trafficking are highly conserved in eukaryotic cells and are responsible for molecular cargo transport and signal transduction among organelles. It remains largely unknown whether clathrin-coated vesicles can be generated from chloroplasts. CHLOROPLAST VESICULATION (CV)-containing vesicles (CVVs) generate from chloroplasts and mediate chloroplast degradation under abiotic stress. In this study, we showed that CV interacted with the clathrin heavy chain (CHC) and induced vesicle budding from the chloroplast inner envelope membrane. Defects on CHC2 and the dynamin-encoding DRP1A gene affected CVV budding and releasing from chloroplast. CHC2 is also required for CV-induced chloroplast degradation and hypersensitivity to water stress. Moreover, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) interacts with CV and impairs the CV-CHC2 interaction. GAPC1 overexpression inhibited CV-mediated chloroplast degradation and hypersensitivity to water stress. CV silencing alleviated the hypersensitivity of gapc1gapc2 plant to water stress. Together, our work revealed a pathway of clathrin-assisted CVV budding from the chloroplast inner envelope membrane, which mediated the stress-induced chloroplast degradation and stress response.

plant biology↗

bioRxiv2021

Multi-omics Analyses Provides Insight into the Biosynthesis Pathways of Fucoxanthin in Isochrysis galbana

Isochrysis galbana is considered an ideal bait for functional foods and nutraceuticals in humans because of its high fucoxanthin (Fx) content. However, multi-omics analysis of the regulation networks for Fx biosynthesis in I. galbana has not been reported. In this study, we report a high-quality genome sequence of I. galbana LG007, which has a 92.73 Mb genome size, with a contig N50 of 6.99 Mb and 14,900 protein-coding genes. Phylogenomic inferences confirmed the monophyly of Haptophyta, with I. galbana sister to Emiliania huxleyi and Chrysochromulina tobinii. Evolutionary analysis revealed an estimated divergence time between I. galbana and E. huxleyi of ~ 133 million years ago (Mya). Gene family analysis indicated that lipid metabolism-related genes exhibited significant expansion, including IgPLMT, IgOAR1 and {Delta}-4 desaturase. Metabolome analysis showed that the content of carotenoid in I. galbana cultured under green light for 7 days was higher than that of white light, and {beta}-carotene was the main carotenoids, accounting for 79.09% of the total carotenoids. Comprehensive analysis of multi-omics analysis revealed that {beta}-carotene, antheraxanthin, zeaxanthin, and Fx content was increased by green light induction, which was significantly correlated with the expression of IgMYB98, IgZDS, IgPDS, IgLHCX2, IgZEP, IgLCYb, and IgNSY. These findings contribute to understanding Fx biosynthesis and its regulation, providing a valuable reference for food and pharmaceutical applications.

bioinformatics↗

bioRxiv2021

The RNA-binding protein Orb2 is associated with microcephaly and supports centrosome asymmetry in neural stem cells

To maintain a balance of self-renewal versus neurogenesis, neural stem cells (NSCs) undergo asymmetric cell division along an invariant polarity axis instructed by centrosomes. In the NSCs of the third instar Drosophila larval brain, the interphase centrosomes are defined by marked asymmetries in protein composition and functional activity as microtubule-organizing centers. Here we show that a conserved RNA-binding protein, Orb2, supports NSC centrosome asymmetry by localizing to the cytoplasm, where it promotes robust apical centrosome maturation and transient basal centrosome inactivation, required for centrosome segregation and spindle morphogenesis. Orb2 is required cell autonomously within NSCs to support centrosome asymmetry and maintenance of the stem cell pool. We suggest Orb2 plays opposing roles in centrosome activation and inactivation at the apical versus basal centrosomes respectively, possibly through the translational regulation of multiple mRNAs. Conversely, loss of orb2 manifests in microcephaly independent of Orb2 function in NSCs. Bioinformatics uncovers a significant overlap among RNA targets between Drosophila Orb2 and human CPEB4, consistent with a conserved role for CPEB proteins in centrosome regulation and neurodevelopment.

cell biology↗

bioRxiv2021

Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly

As the microtubule-organizing centers (MTOCs) of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin (PCNT)-like protein (PLP) is a key centrosome component that directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and MTOC function. Here, we investigate the post-transcriptional regulation of plp mRNA. We identify conserved binding sites for cytoplasmic polyadenylation element binding (CPEB) proteins within the plp 3-untranslated region and examine the role of the CPEB ortholog, oo18 RNA-binding protein (Orb), in plp mRNA regulation. Our data show Orb biochemically interacts with plp mRNA and promotes PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and function in PCM scaffolding is compromised in orb mutant embryos, resulting in genome instability and embryonic lethality. Moreover, we find PLP over-expression can restore centrosome scaffolding and rescue the cell division defects caused by orb depletion. Our data suggest Orb modulates PLP expression at the level of plp mRNA polyadenylation and showcases the post-transcriptional regulation of core, conserved centrosomal mRNAs as critical for centrosome function.

cell biology↗

bioRxiv2021

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leveraged proximity proteomics and conducted an siRNA screen to define the functional interactome of EBOV polymerase. As proof-of-principle, we validated two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose both GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduced viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

microbiology↗