Biology subjects

Taylor, J. P.

Publications and source records attributed to Taylor, J. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Competing Molecular Interactions Govern the Dynamical Arrest of G3BP1 Condensates

G3BP1 is a central scaffold of stress granules (SGs). Upon cellular stress, G3BP1 forms complex coacervates with translationally repressed mRNAs and recruits multiple RNA-binding proteins to form reversible biomolecular condensates. Persistent SGs are linked to age-dependent dynamical arrest and impaired disassembly. Here, we employ active and passive nanoscale rheology with optical tweezers to show that G3BP1 condensates evolve from being dominantly viscous fluids to dynamically arrested network glasses characterized by nanoscale caging and elastic memory. Integrating atomistic and coarse-grained simulations with experiments, we find that electrostatic interactions between the oppositely charged intrinsically disordered regions drive condensate ageing. RNA modulates these interactions in a length-and structure-dependent manner, delaying dynamic arrest, whereas Caprin-1 binding to the NTF2L domain has little effect. Together, these findings reveal how competing inter-IDR and IDR-RNA interactions govern condensate ageing and material-state transitions. The findings have broader implications for the regulation of SG dynamics in cells.

biophysics↗

bioRxiv2025

Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RBPs, including G3BP1/2 and FXR1/FXR2/FMR1, but how these RBPs modulate alphaviral replication and whether it would be possible to target these RBPs for antiviral treatment are less explored. Here, using SFV as a model, we report that SFV nsP3 exploits G3BP for its condensation and transforms antiviral stress granules into proviral nsP3-G3BP co-condensates. The gel-like co-condensates of nsP3 and G3BP enrich and protect viral genomic RNAs from host RNase degradation and serve as viral translational hubs to promote viral replication. The mode of nsP3-RBP co-condensation is prevalent across alphaviruses, and disruption of nsP3 condensates is an efficient antiviral approach. Thus, these findings uncover a general anti-alphavirus strategy based on the conserved reliance of nsP3-RBP co-condensation.

cell biology↗

bioRxiv2025

Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

The phase separation of biomolecules into so-called stress granules (SGs) allows the cell to tightly regulate translation activity in response to different stimuli, such as oxidative stress, starvation, or the recognition of non-cellular RNA. Recent reports suggest SGs induced during viral infection, may act as a crossroad between the cellular stress response and the activation of the innate immune response. Here, we aimed to dissect the role of SGs in the context of yellow fever virus (YFV) infection. We found that YFV infection resulted in translational shut-off from 24 hpi on followed by the formation of SGs at 48 hpi, a delay potentially associated with the ability of the YFV capsid to inhibit SG formation, through an interaction with the major SG scaffolding protein G3BP1. To elucidate the role of YFV-induced SGs during infection, we inhibited SG assembly using a small-molecule inhibitor and find that SG formation does not influence viral replication. Uncovering the first proteome of virus-induced SGs, our compositional analysis revealed a specific enrichment of proteins associated with mitochondrial processes in YFV-induced SGs. Indeed, we show that YFV infection results in mitochondrial damage and dysfunction. Together, we propose that YFV-induced SGs may be involved not only in the modulation of cellular homeostasis but also in influencing mitochondrial functions. Author SummaryViruses impose a major burden on the infected host - from structural rearrangements needed to assemble replication complexes, to exploiting cellular energy resources and genetic rewiring associated with antiviral responses. The assembly of membrane-less organelles such as stress granules (SGs) enable cells to rapidly tune cellular processes upon sensing of stresses such as viruses. Moreover, the cells innate immune response is proposed to be regulated by SGs and in turn many viruses disrupt or highjack their components. Yet, the molecular basis for SG functions during infection remain ambiguous. Here we investigated the interplay between yellow fever virus (YFV) infection and SGs. We demonstrate that infection with attenuated or pathogenic viruses result in the formation of SGs. Their compositional analysis reveal that they sequester mitochondrial proteins, correlating with altered mitochondrial functions during infection. This highlights a novel complex interplay between membrane-bound and membrane-less organelles which could present novel opportunities for antiviral therapies.

microbiology↗

bioRxiv2025

Preclinical evaluation of antisense oligonucleotide therapy in a mouse model of HNRNPH2-related neurodevelopmental disorder

Mutations in HNRNPH2 cause an X-linked disorder characterized by developmental delay, intellectual disability, motor and gait disturbances, and seizures. Murine models that reproduce key clinical features of HNRNPH2-related neurodevelopmental disorder suggest that it may result from a toxic gain of function of the mutant protein or a complex loss of normal HNRNPH2 function with impaired compensation by its homolog, HNRNPH1. In this study, we tested gapmer antisense oligonucleotides (ASOs) that target murine Hnrnph2 in a non-allele-specific manner. The lead ASO reduced Hnrnph2 mRNA and protein levels while inducing compensatory upregulation of Hnrnph1 in both WT and Hnrnph2 mutant mouse brains. A single intracerebroventricular injection of the Hnrnph2 ASO into neonatal mutant Hnrnph2 mice rescued molecular and audiogenic seizure phenotypes and improved motor and cognitive functions. ASO treatment at the juvenile stage also rescued audiogenic seizures and motor deficits. In contrast, Hnrnph2 ASO administration did not improve survival, body weight, or hydrocephalus. In human iPSC-derived neurons, a human-specific HNRNPH2 research ASO reduced HNRNPH2 and upregulated HNRNPH1 mRNA levels. Mechanistically, we demonstrate that HNRNPH1 expression is regulated by alternative splicing and that HNRNPH2 modulates this process. These findings provide preclinical proof of concept for HNRNPH2 ASO therapy and offer insights into its underlying molecular mechanism. One Sentence SummaryASO-mediated Hnrnph2 knockdown induces Hnrnph1 upregulation and rescues phenotypes in a mouse model of HNRNPH2-related neurodevelopmental disorder.

animal behavior and cognition↗

bioRxiv2025

Integrative multiomic analysis links TDP-43-driven splicing defects to cascading proteomic disruption of ALS/FTD pathways

Loss of nuclear TDP-43 is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is known to regulate RNA processing, including repression of cryptic exons, we currently lack a systems-level understanding of the consequences of TDP-43 loss. To address this, we generated multiomic datasets, including RNA-seq and proteomics, from human iPSC-derived neurons depleted of TDP-43. We found that differentially spliced genes, many expressing cryptic exons, had the greatest protein reductions. Surprisingly, nearly half of differentially expressed proteins were neither mis-spliced, nor differentially expressed genes; most of these also had no reported mis-splicing in seven additional post-mortem and iPSC-derived neuron datasets. Integrative network analysis identified a high-confidence disease-specific subnetwork of over 700 interacting proteins, enriched for mRNA processing, synaptic function, and autophagy. Comparison with post-mortem ALS and FTD samples revealed convergent protein and pathway disruptions. We experimentally validated network-predicted effects of cryptic splicing in ATG4B, STMN2, and DAPK1. Our analyses reveal new TDP-43-dependent molecular cascades and nominate central genes as potential ALS/FTD therapeutic targets.

neuroscience↗

bioRxiv2025

A point mutation in the nuclear speckle protein and splicing factor SRRM2 is associated with amyotrophic lateral sclerosis and causes dysregulation of synapse-associated genes

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive loss of motor neuron function. ALS is a multifactorial disease which can originate from complex genetic and environmental factors. The identification of risk factors and their molecular contribution to ALS expand our understanding of the disease mechanism. Here, we describe a family with dominantly inherited degeneration, which carries a mutation in the serine/arginine repetitive matrix 2 gene (SRRM2). SRRM2 is essential for nuclear speckle formation and a constitutive member of the RNA splicing machinery. To investigate how the mutation in SRRM2 contributed to the ALS pathogenesis, we examined its effect on a model cell line, where the point mutation was introduced in the endogenous gene. Surprisingly, we found that the resulting single amino acid exchange led to the loss of one protein-protein interaction, between SRRM2 and the splicing factor ACIN1. Transcriptome studies further revealed wide-spread differential gene expression, which converged on the dysregulation of synapse-associated pathways. Together, our findings identify SRRM2 as a novel ALS risk factor and provide mechanistic insights into how its mutation can be linked to ALS pathology.

molecular biology↗

bioRxiv2024

Coxsackievirus B infection invokes unique cell-type specific responses in primary human pancreatic islets

Coxsackievirus B (CVB) infection has long been considered an environmental factor precipitating Type 1 diabetes (T1D), an autoimmune disease marked by loss of insulin-producing {beta} cells within pancreatic islets. Previous studies have shown CVB infection negatively impacts islet function and viability but do not report on how virus infection individually affects the multiple cell types present in human primary islets. Therefore, we hypothesized that the various islet cell populations have unique transcriptional responses to CVB infection. Here, we performed single-cell RNA sequencing on human cadaveric islets treated with either CVB or poly(I:C), a viral mimic, for 24 and 48 hours. Our global analysis reveals CVB differentially induces dynamic transcriptional changes associated with multiple cell processes and functions over time whereas poly(I:C) promotes an immune response that progressively increases with treatment duration. At the single-cell resolution, we find CVB infects all islet cell types at similar rates yet induces unique cell-type specific transcriptional responses with {beta}, , and ductal cells having the strongest response. Sequencing and functional data suggest that CVB negatively impacts mitochondrial respiration and morphology in distinct ways in {beta} and cells, while also promoting the generation of reactive oxygen species. We also observe an increase in the expression of the long-noncoding RNA MIR7-3HG in {beta} cells with high viral titers and reveal its knockdown reduces gene expression of viral proteins as well as apoptosis in stem cell-derived islets. Together, these findings demonstrate a cell-specific transcriptional, temporal, and functional response to CVB infection and provide new insights into the relationship between CVB infection and T1D.

cell biology↗

bioRxiv2024

Metastable condensates suppress conversion to amyloid fibrils

Stress granules form via co-condensation of RNA-binding proteins containing prion-like low complexity domains (PLCDs) with RNA molecules. Homotypic interactions among PLCDs can drive amyloid fibril formation that is enhanced by ALS-associated mutations. We report that condensation-versus fibril-driving homotypic interactions are separable for A1-LCD, the PLCD of hnRNPA1. Separable interactions lead to thermodynamically metastable condensates and globally stable fibrils. Interiors of condensates suppress fibril formation whereas interfaces have the opposite effect. ALS-associated mutations enhance the stability of fibrils and weaken condensate metastability, thus enhancing the rate of fibril formation. We designed mutations to enhance A1-LCD condensate metastability and discovered that stress granule disassembly in cells can be restored even when the designed variants carry ALS-causing mutations. Therefore, fibril formation can be suppressed by condensate interiors that function as sinks. Condensate sink potentials are influenced by their metastability, which is tunable through separable interactions even among minority components of stress granules.

biophysics↗

bioRxiv2023

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

cell biology↗

bioRxiv2023

Identification of small molecule inhibitors of G3BP-driven stress granule formation

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

cell biology↗

bioRxiv2023

Dynamic mapping of proteome trafficking within and between living cells by TransitID

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules, uncovering a role for stress granules in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID introduces a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

cell biology↗

bioRxiv2023

A new Karyopherin-β2 binding PY-NLS epitope of HNRNPH2 is linked to neurodevelopmental disorders

The normally nuclear HNRNPH2 is mutated in HNRNPH2-related X-linked neurodevelopmental disorder causing the protein to accumulate in the cytoplasm. Interactions of HNRNPH2 with its importin Karyopherin-{beta}2 (Transportin-1) had not been studied. We present a structure that shows Karyopherin-{beta}2 binding HNRNPH2 residues 204-215, a proline-tyrosine nuclear localization signal or PY-NLS that contains a typical R-X2-4-P-Y motif, 206RPGPY210, followed a new Karyopherin-{beta}2 binding epitope at 211DRP213 that make many interactions with Karyopherin-{beta}2 W373. Mutations at each of these sites decrease Karyopherin-{beta}2 binding affinities by 70-100 fold, explaining aberrant accumulation in cells and emphasizing the role of nuclear import defects in the disease. Sequence/structure analysis suggests that the new epitope C-terminal of the PY-motif, which binds Karyopherin-{beta}2 W373, is rare and thus far limited to close paralogs HNRNPH2, HNRNPH1 and HNRNPF. Karyopherin-{beta}2 W373, a HNRNPH2-binding hotspot, corresponds to W370 of close paralog Transportin-2, a site of pathological variants in patients with neurodevelopmental abnormalities, suggesting that Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities. SummaryHNRNPH2 variants in HNRNPH2-related X-linked neurodevelopmental disorder aberrantly accumulate in the cytoplasm. A structure of Karyopherin-{beta}2*HNRNPH2 explains nuclear import defects of the variants, reveals a new NLS epitope that suggests mechanistic changes in pathological variants of Karyopherin-{beta}2 paralog Transportin-2.

biophysics↗

bioRxiv2022

A murine model of hnRNPH2-related neurodevelopmental disorder recapitulates clinical features of human disease and reveals a mechanism for genetic compensation of HNRNPH2

Mutations in HNRNPH2 cause an X-linked neurodevelopmental disorder with a phenotypic spectrum that includes developmental delay, intellectual disability, language impairment, motor function deficits, and seizures. More than 90% of patients with this disorder have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2, although the specific pathogenic consequences of these mutations have not been examined. Here we found that hnRNPH2 NLS mutations result in reduced interaction with the nuclear transport receptor Kap{beta}2 in vitro and in cultured human cells. These mutations also cause modest accumulation of hnRNPH2 in the cytoplasm, suggesting that mislocalization of the protein might contribute to pathogenesis. We generated two knock-in mouse models with human-equivalent mutations in the endogenous mouse gene Hnrnph2, as well as Hnrnph2 knockout (KO) mice, and subjected them to extensive phenotyping. Mutant knock-in mice displayed a spectrum of phenotypes that recapitulated aspects of the human disorder, including reduced survival in males, craniofacial abnormalities, impaired motor and cognitive functions, and increased susceptibility to audiogenic seizures. Mutant knock-in male mice developed more severe phenotypes than female mice, likely due to differences in X-chromosome gene dosage. In contrast, two independent lines of Hnrnph2 KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a close paralog of Hnrnph2, whereas mutant Hnrnph2 knock-in mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 might be sufficient to counteract the loss of hnRNPH2. These findings suggest that the pathogenesis of HNRNPH2-related disorder in humans may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The mutant knock-in mice described here are an important resource for preclinical studies to assess the potential benefit of either gene replacement or therapeutic knockdown of mutant hnRNPH2.

neuroscience↗

bioRxiv2021

MAP4K3 inhibits Sirtuin-1 to repress LKB1-AMPK to promote amino acid dependent activation of mTORC1

mTORC1 is the key rheostat controlling cellular metabolic state. Of the various inputs to mTORC1, the most potent effector of intracellular nutrient status is amino acid supply. Despite an established role for MAP4K3 in promoting mTORC1 activation in the presence of amino acids, the signaling pathway by which MAP4K3 controls mTORC1 activation remains unknown. Here we examined the process of MAP4K3 activation of mTORC1 and found that MAP4K3 represses the LKB1-AMPK pathway to prevent TSC1/2 complex inactivation of Rheb. When we sought the regulatory link between MAP4K3 and LKB1 inhibition, we discovered that MAP4K3 physically interacts with the master nutrient regulatory factor Sirtuin-1 and phosphorylates Sirtuin-1 to repress LKB1 activation. Our results reveal the existence of a novel signaling pathway linking amino acid satiety with MAP4K3-dependent suppression of SIRT1 to inactivate the repressive LKB1-AMPK pathway and thereby potently activate the mTORC1 complex to dictate the metabolic disposition of the cell.

cell biology↗

bioRxiv2021

Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes

Type 1 diabetes is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of {beta}-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individuals collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and {beta}-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

immunology↗

bioRxiv2021

Ubiquitination of G3BP1 mediates stress granule disassembly in a stress-specific manner

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in response to a variety of stressors and are normally disassembled after stress is removed. Whereas the composition of stress granules and the mechanisms underlying their assembly have been extensively studied, far less is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases. Here we report that stress granule disassembly is context-dependent and, in the setting of heat shock, requires ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. Ubiquitinated G3BP1 interacts with the ER-resident protein FAF2, which engages the ubiquitin-dependent segregase p97/VCP. Targeting G3BP1 enables the stress granule-specific interaction network to fall below the percolation threshold for phase separation, which causes disassembly. One Sentence SummaryUbiquitination of G3BP1 mediates FAF2- and p97/VCP-dependent disassembly of heat-induced stress granules

cell biology↗

bioRxiv2021

Ubiquitination is essential for recovery of cellular activities followingheat shock

Eukaryotic cells respond to stress via adaptive programs that include reversible shutdown of key cellular processes, the formation of stress granules, and a global increase in ubiquitination. The primary function of this ubiquitination is generally considered to be tagging damaged or misfolded proteins for degradation. Here we show that different types of stress generate distinct ubiquitination patterns. For heat stress, ubiquitination correlates with cellular activities that are downregulated during stress, including nucleocytoplasmic transport and translation, as well as with stress granule constituents. Ubiquitination is not required for the shutdown of these processes or for stress granule formation, but is essential for resumption of cellular activities and for stress granule disassembly. These findings indicate that stress-induced ubiquitination primes the cell for recovery following heat stress. One Sentence SummaryStress-induced ubiquitination is essential for recovery of cellular activities following heat stress.

cell biology↗

bioRxiv2020

High fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate

Liquid-liquid phase separation (LLPS) is an important mechanism of intracellular organization that underlies the assembly of a variety of distinct RNP granules. Fundamental biophysical principles governing LLPS during RNP granule assembly have been revealed by simple in vitro systems consisting of several components, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in live cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells, and allows for a wide variety of studies of membraneless organelles.

cell biology↗