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Cells use transient membraneless organelles to regulate biological reaction networks. For example, stress granules selectively store mRNA to downregulate protein expression in response to heat or oxidative stress. Models mimicking this active behavior should be established to better understand in vivo regulation involving compartmentalization. Here we use active, complex coacervate droplets as a model for membraneless organelles to spatiotemporally control the activity of a catalytic DNA (DNAzyme). Upon partitioning into these peptide‐RNA droplets, the DNAzyme unfolds and loses its ability to catalyze the cleavage of a nucleic acid strand. We can transiently pause the DNAzyme activity upon inducing droplet formation with fuel. After fuel consumption, the DNAzyme activity autonomously restarts. We envision this system could be used to up and downregulate multiple reactions in a network, helping understand the complexity of a cell's pathways. By creating a network where the DNAzyme could reciprocally regulate the droplet properties, we would have a powerful tool for engineering synthetic cells. [ABSTRACT FROM AUTHOR]

The C. elegans Argonaute protein PRG‐1/Piwi and associated piRNAs protect metazoan genomes by silencing transposons and other types of foreign DNA. As prg‐1 mutants are propagated, their fertility deteriorates prior to the onset of a reproductive arrest phenotype that resembles a starvation‐induced stress response. We found that late‐generation prg‐1 mutants with substantially reduced fertility were long‐lived, whereas early‐ or mid‐generation prg‐1 mutants had normal lifespans. Loss of the stress response transcription factor DAF‐16 caused mid‐ or late‐generation prg‐1 mutants to live very short lives, whereas overexpression of DAF‐16 enabled both mid‐ and late‐generation prg‐1 mutants to live long. Cytoplasmic P‐bodies that respond to stress increased in long‐lived late‐generation prg‐1 mutants and were transmitted to F1 but not F2 cross‐progeny. Moreover, moderate levels of heritable stress shorten late‐generation prg‐1 mutant longevity when DAF‐16 or P bodies are deficient. Together, these results suggest that the longevity of late‐generation prg‐1 mutants is a hormetic stress response. However, dauer larvae that occur in response to stress were not observed in late‐generation prg‐1 mutants. Small germ cell nucleoli that depended on germline DAF‐16 were present in late‐generation prg‐1 mutants but were not necessary for their longevity. We propose that prg‐1 mutant germ cells transmit a form of heritable stress, high levels of which promote longevity and strongly reduce fertility. The heritable stress transmitted by prg‐1/Piwi mutant germ cells may be generally relevant to epigenetic inheritance of longevity. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2, a highly contagious coronavirus, is responsible for the global pandemic of COVID-19 in 2019. Currently, it remains uncertain whether SARS-CoV-2 possesses oncogenic or oncolytic potential in influencing tumor progression. Therefore, it is important to evaluate the clinical and functional role of SARS-CoV-2 on tumor progression. Methods: Here, we integrated bioinformatic analysis of COVID-19 RNA-seq data from the GEO database and performed functional studies to explore the regulatory role of SARS-CoV-2 in solid tumor progression, including lung, colon, kidney and liver cancer. Results: Our results demonstrate that infection with SARS-CoV-2 is associated with a decreased expression of genes associated with cancer proliferation and metastasis in lung tissues from patients diagnosed with COVID-19. Several cancer proliferation or metastasis related genes were frequently downregulated in SARS-CoV-2 infected intestinal organoids and human colon carcinoma cells. In vivo and in vitro studies revealed that SARS-CoV-2 nucleocapsid (N) protein inhibits colon and kidney tumor growth and metastasis through the N-terminal (NTD) and the C-terminal domain (CTD). The molecular mechanism indicates that the N protein of SARS-CoV-2 interacts with YBX1, resulting in the recruitment of PKM mRNA into stress granules mediated by G3BP1. This process ultimately destabilizes PKM expression and suppresses glycolysis. Conclusion: Our study reveals a new function of SARS-CoV-2 nucleocapsid protein on tumor progression. [ABSTRACT FROM AUTHOR]

Stress granules (SG) are membraneless ribonucleoprotein-based cytoplasmic organelles that assemble in response to stress. Their formation is often associated with an almost global suppression of translation, and the aberrant assembly or disassembly of these granules has pathological implications in neurodegeneration and cancer. In cancer, and particularly in the presence of oncogenic KRAS mutations, in vivo studies concluded that SG increase the resistance of cancer cells to stress. Hence, SG have recently been considered a promising target for therapy. Here, starting from our observations that genes coding for SG proteins are stimulated during development of pancreatic ductal adenocarcinoma, we analyze the formation of SG during tumorigenesis. We resort to in vitro, in vivo and in silico approaches, using mouse models, human samples and human data. Our analyses do not support that SG are formed during tumorigenesis of KRAS-driven cancers, at least that their presence is not universal, leading us to propose that caution is required before considering SG as therapeutic targets. Synopsis: Despite higher levels of mRNA encoding SG proteins during pancreatic tumorigenesis, this study finds no SG formation in Kras-driven cancers. This suggests that SG may not be a universal therapeutic cancer target. Stress granules (SG) were not detected in Kras-driven lesions in mouse models or in human PDAC samples. The protein interactome of G3BP does not indicate a role for G3BP related to SG formation. SG are not universally present or relevant in Kras-mutant cancers. Caution is warranted before pursuing SG as a cancer therapeutic target in Kras-mutant cancers. Despite higher levels of mRNA encoding SG proteins during pancreatic tumorigenesis, this study finds no SG formation in Kras-driven cancers. This suggests that SG may not be a universal therapeutic cancer target. [ABSTRACT FROM AUTHOR]

The role of stress granules (SGs) in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that SG formation contributes to abnormal vascular phenotypes, and cardiac and skeletal muscle dysfunction in PAH. Using the rat Sugen/hypoxia (SU/Hx) model of PAH, we demonstrate the formation of SG puncta and increased expression of SG proteins compared to control animals in lungs, right ventricles, and soleus muscles. Acetazolamide (ACTZ) treatment ameliorated the disease and reduced SG formation in all of these tissues. Primary pulmonary artery smooth muscle cells (PASMCs) from diseased animals had increased SG protein expression and SG number after acute oxidative stress and this was ameliorated by ACTZ. Pharmacologic inhibition of SG formation or genetic ablation of the SG assembly protein (G3BP1) altered the SU/Hx-PASMC phenotype by decreasing proliferation, increasing apoptosis and modulating synthetic and contractile marker expression. In human PAH lungs, we found increased SG puncta in pulmonary arteries compared to control lungs and in human PAH-PASMCs we found increased SGs after acute oxidative stress compared to healthy PASMCs. Genetic ablation of G3BP1 in human PAH-PASMCs resulted in a phenotypic switch to a less synthetic and more contractile phenotype. We conclude that increased SG formation in PASMCs and other tissues may contribute to PAH pathogenesis. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are stress-induced RNA condensates consisting of stalled initiation complexes resulting from translational inhibition. The biochemical composition and function of SGs are highly diverse, and this diversity has been attributed to different stress conditions, signalling pathways involved and specific cell types. Interestingly, mRNA decay components, which are found in ubiquitous cytoplasmic foci known as processing bodies (PB), have also been identified in SG proteomes. A major challenge in current SG studies is to understand the cause of SG diversity, as well as the function of SG under different stress conditions. Trypanosoma brucei is a single-cellular parasite that causes Human African Trypanosomiasis (sleping sickness). In this study, we showed that by varying the supply of extracellular carbon sources during starvation, cellular ATP levels changed rapidly, resulting in SGs of different compositions and dynamics. We identified a subset of SG components, which dissociated from the SGs in response to cellular ATP depletion. Using expansion microscopy, we observed sub-granular compartmentalization of PB- and SG-components within the stress granules. Our results highlight the importance of cellular ATP in SG composition and dynamics, providing functional insight to SGs formed under different stress conditions. Author summary: Stress granules (SGs) and processing bodies (PBs) are cytoplasmic RNA granules containing multiple proteins with functions in translation initiation and mRNA degradation. mRNA and proteins can move between SGs, PBs and translating polysomes, likely regulating gene expression under different cellular conditions. However, little is known about the cellular factors regulating the dynamic behaviour of these RNA granules. In the unicellular Trypanosome parasites, SGs are formed under starvation stress and during differentiation. In this study, we found that in starved T. brucei, the composition and the dynamics of the SGs are distinct, depending on the supply of extracellular carbon sources and subsequent cellular energy (ATP) levels. Using expansion microscopy and super-resolution microscopy, functionally-distinct protein components are found in different sub-granular compartments within the SGs. Our findings emphasized the dynamic changes in SG composition, organization, and potential functions, which are affected by cellular ATP levels that can fluctuate under various stress conditions. [ABSTRACT FROM AUTHOR]

Physical properties of biomolecular condensates formed via phase separation of proteins and nucleic acids are associated with cell physiology and disease. Condensate properties can be regulated by several cellular factors including post-translational modifications. Here, we introduce an application of intermolecular energy migration via homo-FRET (Förster resonance energy transfer), a nanometric proximity ruler, to study the modulation in short- and long-range protein-protein interactions leading to the changes in the physical properties of condensates of fluorescently-tagged FUS (Fused in Sarcoma) that is associated with the formation of cytoplasmic and nuclear membraneless organelles. We show that homoFRET captures modulations in condensate properties of FUS by RNA, ATP, and post-translational arginine methylation. We also extend the homoFRET methodology to study the in-situ formation of cytoplasmic stress granules in mammalian cells. Our studies highlight the broad applicability of homoFRET as a potent generic tool for studying intracellular phase transitions involved in function and disease. The properties of biomolecular condensates can be regulated by multiple factors, including intermolecular dynamics. Here, the authors use fluorescence anisotropy-based homoFRET imaging to monitor the intermolecular interactions and supramolecular packing that underlie the modulation of biomolecular condensate properties. [ABSTRACT FROM AUTHOR]

Protein aggregation plays key roles in age-related degenerative diseases, but how different proteins coalesce to form inclusions that vary in composition, morphology, molecular dynamics and confer physiological consequences is poorly understood. Here we employ a general reporter based on mutant Hsp104 to identify proteins forming aggregates in human cells under common proteotoxic stress. We identify over 300 proteins that form different inclusions containing subsets of aggregating proteins. In particular, TDP43, implicated in Amyotrophic Lateral Sclerosis (ALS), partitions dynamically between two distinct types of aggregates: stress granule and a previously unknown non-dynamic (solid-like) inclusion at the ER exit sites (ERES). TDP43-ERES co-aggregation is induced by diverse proteotoxic stresses and observed in the motor neurons of ALS patients. Such aggregation causes retention of secretory cargos at ERES and therefore delays ER-to-Golgi transport, providing a link between TDP43 aggregation and compromised cellular function in ALS patients. Protein aggregation has been implicated in several neurodegenerative diseases. Here, Wu et al. utilized a general aggregate reporter to identify aggregation-prone proteins and discover that TDP43 aggregates at ER-exit sites (ERES) under proteotoxic stress and impairs ER-to-Golgi transport, linking TDP43 aggregation and ER dysfunction. [ABSTRACT FROM AUTHOR]

The integrated stress response, especially stress granules (SGs), contributes to host immunity. Typical G3BP1+ stress granules (tSGs) are usually formed after virus infection to restrain viral replication and stimulate innate immunity. Recently, several SG-like foci or atypical SGs (aSGs) with proviral function have been found during viral infection. We have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein induces atypical N+/G3BP1+ foci (N+foci), leading to the inhibition of host immunity and facilitation of viral infection. However, the precise mechanism has not been well clarified yet. In this study, we showed that the SARS-CoV-2 N (SARS2-N) protein inhibits dsRNA-induced growth arrest and DNA damage-inducible 34 (GADD34) expression. Mechanistically, the SARS2-N protein promotes the interaction between GADD34 mRNA and G3BP1, sequestering GADD34 mRNA into the N+foci. Importantly, we found that GADD34 participates in IRF3 nuclear translocation through its KVRF motif and promotes the transcription of downstream interferon genes. The suppression of GADD34 expression by the SARS2-N protein impairs the nuclear localization of IRF3 and compromises the host's innate immune response, which facilitates viral replication. Taking these findings together, our study revealed a novel mechanism by which the SARS2-N protein antagonized the GADD34-mediated innate immune pathway via induction of N+foci. We think this is a critical strategy for viral pathogenesis and has potential therapeutic implications. [ABSTRACT FROM AUTHOR]

Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment. This study reveals how the intrinsically disordered region of eIF4B governs its transition from monomers to oligomers and condensed droplets, providing key insights into how its self-association may be regulated to carry out its cellular functions. [ABSTRACT FROM AUTHOR]

Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA–protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG–PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L’s role. [ABSTRACT FROM AUTHOR]

Cellular stress and ageing involve an increase in crowding and aggregation of amylogenic proteins. We here investigate if crowding is the intrinsic cause of aggregation and utilise a previously established non-protein aggregation sensor, namely pseudoisocyanine chloride (PIC). PIC shows fibrillization in cells into a highly fluorescent J-aggregated state and is sensitive to crowding. Surprisingly, cell stress conditions stabilise the monomeric rather than the aggregated state of PIC both in the cytoplasm and in stress granules. Regarding the different physiochemical changes of the cytoplasm occurring upon cell stress, involving volume reduction, phase separation and solidification, the intrinsic crowding effect is not the key factor to drive associated self-assembly processes. Cellular stress and ageing involve an increase in crowding and aggregation of amylogenic proteins, but the connection between protein destabilisation and the onset of aggregation is poorly understood. Here, the authors utilize a non-protein aggregation sensor based on pseudoisocyanine chloride to analyse the effect of macromolecular crowding in the cytoplasm on the self-assembly process, and find that the high crowding densities observed in the cytoplasm and stress granules upon stress are not an intrinsic cause for aggregation of amylogenic proteins. [ABSTRACT FROM AUTHOR]

Introduction: Retinoic acid (RA) was first recognised to be important for the central nervous system (CNS) in its developmental regulatory role and, given this action, it has been proposed in the adult CNS to regulate plasticity and promote regeneration. These types of roles have included support of neurogenesis, induction of neurite outgrowth, and protection from neuronal death. These functions are predominantlymediated by the retinoic acid receptor (RAR) transcription factor, and hence agonists for the RARs have been tested in a variety of models of neurodegeneration. This present study employs several in vitro models less explored for the action of RAR agonists to reverse neurodegeneration. Methods: A series of assays are used in which neuronal cells are placed under the types of stress that have been linked to neurodegeneration, in particular amyotrophic lateral sclerosis (ALS), and the neuroprotective influence of a new potent agonist for RAR, ellorarxine, is tested out. In these assays, neuronal cells were subjected to excitotoxic stress induced by glutamate, proteostasis disruption caused by epoxomicin, and oxidative stress leading to stress granule formation triggered by sodium arsenite. Results: Ellorarxine effectively reversed neuronal death in excitotoxic and proteostasis disruption assays andmitigated stress granule formation induced by sodiumarsenite. This study also highlights for the first time the novel observation of RAR modulation of stress granules, although it is unknown whether this change in stress granules will be neuroprotective or potentially regenerative. Furthermore, the distribution of RAR agonists following intraperitoneal injection was assessed in mice, revealing preferential accumulation in the central nervous system, particularly in the spinal cord, compared to the liver. Gene expression studies in the spinal cord demonstrated that ellorarxine induces transcriptional changes at a low dose (0.01 mg/kg). Discussion: These findings underscore the therapeutic potential of RAR agonists, such as ellorarxine, for ALS and potentially other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The chirality of a chemical differentiates it from its mirror-image counterpart. This unique property has significant implications in chemistry, biology, and drug discovery, where chiral chemicals display high selectivity and activity in achieving target specificity and reducing attrition rates in drug development. Stress granules (SGs) are dynamic assemblies of proteins and RNA that form in the cytoplasm of cells under stress conditions. Modulating their formation or disassembly could offer a novel approach to treating a wide range of diseases. This has led to significant interest in SGs as potential therapeutic targets. This study examined the NTF2-like domain of G3BP1 as a possible target for SG modulation. Molecular docking was used to simulate the interactions of compounds with the domain, and a potential candidate with a chiral structure was identified. The experiments showed that the compound induced the formation of SG-like granules. Importantly, the ability of this compound to modulate SG offers valuable insights into a new mechanism underlying the dynamics and promoting the assembly of SGs, and this new mechanism, in turn, holds potential for the development of drugs with diverse mechanisms of action and potentially synergistic effects. [ABSTRACT FROM AUTHOR]

Molecular chaperones play a central role in protein disaggregation. However, the molecular determinants that regulate this process are poorly understood. Hsp104 is an AAA+ ATPase that disassembles stress granules and amyloids in yeast through collaboration with Hsp70 and Hsp40. In vitro studies show that Hsp104 processes different types of protein aggregates by partially translocating or threading polypeptides through the central pore of the hexamer. However, it is unclear how Hsp104 processing influences client protein function in vivo. The middle domain (MD) of Hsp104 regulates ATPase activity and interactions with Hsp70. Here, we tested how MD variants, Hsp104A503S and Hsp104A503V, process different protein aggregates. We establish that engineered MD variants fail to resolve stress granules but retain prion fragmentation activity required for prion propagation. Using the Sup35 prion protein, our in vitro and in vivo data indicate that the MD variants can disassemble Sup35 aggregates, but the disaggregated protein has reduced GTPase and translation termination activity. These results suggest that the middle domain can play a role in sensing certain substrates and plays an essential role in ensuring the processed protein is functional. Author summary: Protein misfolding and assembly can lead to the formation highly ordered aggregates called amyloid. Amyloid is associated with several proteinopathies, including Alzheimer's and Parkinson's disease. Hsp104, the major chaperone disaggregase in yeast, fragments amyloid associated with yeast prions by working in concert with Hsp70 and Hsp40 chaperones to extract monomers from the amyloid in a partial-threading mechanism. Engineered variants within the middle domain (MD) of Hsp104, Hsp104A503S and Hsp104A503V, effectively disaggregate and reduce toxicity associated with human aggregating proteins. However, how the middle domain influences protein processing is not clearly defined. Our in vitro and in vivo data suggest that the MD can help ensure that disaggregated proteins are functional immediately after processing. [ABSTRACT FROM AUTHOR]

RNAs and RNA-binding proteins can undergo spontaneous or active condensation into phase-separated liquid-like droplets. These condensates are cellular hubs for various physiological processes, and their dysregulation leads to diseases. Although RNAs are core components of many cellular condensates, the underlying molecular determinants for the formation, regulation, and function of ribonucleoprotein condensates have largely been studied from a protein-centric perspective. Here, we highlight recent developments in ribonucleoprotein condensate biology with a particular emphasis on RNA-driven phase transitions. We also present emerging future directions that might shed light on the role of RNA condensates in spatiotemporal regulation of cellular processes and inspire bioengineering of RNA-based therapeutics. Ribonucleoprotein granules are thought to form via phase separation of RNA and associated proteins. Wadsworth et al. discuss the distinctive ways in which RNA critically contributes to the formation, stability, spatiotemporal dynamics, and function of ribonucleoprotein granules, highlighting emerging perspectives in RNA biology and disease processes. [ABSTRACT FROM AUTHOR]

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress. [Display omitted] • RBP-centric RNA-aware PPI network of the mRNA life cycle in human cells • Life-cycle stages assigned to network proteins, with ∼50% linked to multiple stages • Highly connected ERH affects nuclear speckle organization and mRNA export • SNRNP200 interacts with stress granules and has distinct cytoplasmic RNA occupancy Street, Rothamel, Brannan, et al. generate an RNA-aware RBP protein-protein interactome across the mRNA life cycle in human cells, leading to the discovery of previously unknown RBP interactions and functions. Investigation into the functions of the two most-connected proteins, ERH and SNRNP200, reveals the utility of the network for multifunctional RBP discovery. [ABSTRACT FROM AUTHOR]

• Generation of bi- and tri-partite murine iPSC-derived neurospheroids. • Induction of inflammatory stress in bi- and tri-partite neurospheroids. • Single and combined transcriptomic and proteomic analyses. • The presence of microglia exacerbates cellular stress in tri-partite neurospheroids. induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX 3 CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1β + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With Type-I interferon (IFN) signalling being an important first line cellular defence mechanism against VZV infection by the peripheral tissues, we here investigated the triggering of innate immune responses in a human neurallike environment. For this, we established and characterised 5-month matured hiPSC-derived neurospheroids (NSPHs) containing neurons and astrocytes. Subsequently, NSPHs were infected with reporter strains of VZV (VZVeGFPORF23) or Sendai virus (SeVeGFP), with the latter serving as an immune-activating positive control. Live cell and immunocytochemical analyses demonstrated VZVeGFP-ORF23 infection throughout the NSPHs, while SeVeGFP infection was limited to the outer NSPH border. Next, NanoString digital transcriptomics revealed that SeVeGFP-infected NSPHs activated a clear Type-I IFN response, while this was not the case in VZVeGFP-ORF23-infected NSPHs. Moreover, the latter displayed a strong suppression of genes related to IFN signalling and antigen presentation, as further demonstrated by suppression of IL-6 and CXCL10 production, failure to upregulate Type-I IFN activated anti-viral proteins (Mx1, IFIT2 and ISG15), as well as reduced expression of CD74, a key-protein in the MHC class II antigen presentation pathway. Finally, even though VZVeGFP-ORF23- infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules upon long-term infection, as well as disruption of cellular integrity within the infected NSPHs. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, and have the capacity to recapitulate the strong immune evasive behaviour towards VZV. [ABSTRACT FROM AUTHOR]

Despite docetaxel combined with cisplatin and 5‐fluorouracil (TPF) being the established treatment for advanced nasopharyngeal carcinoma (NPC), there are patients who do not respond positively to this form of therapy. However, the mechanisms underlying this lack of benefit remain unclear. DCAF7 is identified as a chemoresistance gene attenuating the response to TPF therapy in NPC patients. DCAF7 promotes the cisplatin resistance and metastasis of NPC cells in vitro and in vivo. Mechanistically, DCAF7 serves as a scaffold protein that facilitates the interaction between USP10 and G3BP1, leading to the elimination of K48‐linked ubiquitin moieties from Lys76 of G3BP1. This process helps prevent the degradation of G3BP1 via the ubiquitin‒proteasome pathway and promotes the formation of stress granule (SG)‐like structures. Moreover, knockdown of G3BP1 successfully reversed the formation of SG‐like structures and the oncogenic effects of DCAF7. Significantly, NPC patients with increased levels of DCAF7 showed a high risk of metastasis, and elevated DCAF7 levels are linked to an unfavorable prognosis. The study reveals DCAF7 as a crucial gene for cisplatin resistance and offers further understanding of how chemoresistance develops in NPC. The DCAF7‐USP10‐G3BP1 axis contains potential targets and biomarkers for NPC treatment. [ABSTRACT FROM AUTHOR]

Anxiety disorder is a universal disease related to neuro-inflammation. Solanesol has shown positive effects because of its anti-inflammatory, anti-tumor, and anti-ulcer properties. This study focused on determining whether solanesol could ameliorate anxiety-like behaviors in a mouse model of neuro-inflammation and identify its working targets. Complete Freund's adjuvant (CFA)-induced mice that were intra-peritoneally administered with solanesol (50 mg/kg) for 1 week showed a statistically significant reduction in anxiety-like behaviors, as measured by open field and elevated plus-maze tests. Western blot analysis revealed that CFA-induced upregulation of the levels of pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor α (TNF-α), which played crucial roles in regulating anxiety, returned to normal in the anterior cingulate cortex (ACC) after solanesol treatment. The level of T cell-restricted intracellular antigen-1 (TIA1), a key component of stress granules, also decreased in the ACC. Moreover, immunofluorescence results indicated that solanesol suppressed CFA-induced microglial and astrocytic activation in the ACC. CFA was injected in the hind paws of TIA1Nestin conditional knockout (cKO) mice to confirm whether TIA1 is a potential modulatory molecule that influences pro-inflammatory cytokines and anxiety-like behaviors. Anxiety-like behaviors could not be observed in cKO mice after CFA injection with IL-1β and TNF-α levels not remarkedly increasing. Our findings suggest that solanesol inhibits neuro-inflammation by decreasing the TIA1 level to reduce IL-1β and TNF-α expression, meanwhile inhibiting microglial and astrocytic activation in the ACC and ultimately ameliorating anxiety-like behaviors in mice. [ABSTRACT FROM AUTHOR]

Summary: Cytoplasmic ribonucleoprotein (RNP) granules are membraneless structures composed of various RNAs and proteins that play important roles in post‐transcriptional regulation. While RNP granules are known to regulate the meiotic entry in some organisms, little is known about their roles in plants.In this study, we observed the cytoplasmic granular structures of rice RNA‐binding protein MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2), which contributes to the control of meiotic entry timing, in leaf protoplasts and spore mother cells. We performed colocalization analysis with known cytoplasmic RNP factors, and domain deletion analysis to assess their impact on granule formation and meiosis progression. Conservation of MEL2 domains across plant species was also explored.Our results indicated that MEL2 granules colocalized with processing body and stress granule factors. The maintenance of granule properties modulated by LOTUS domain and the intrinsically disordered region (IDR) is essential for proper MEL2 function in meiosis progression. MEL2‐like proteins widely found in plant kingdom conserved LOTUS domain followed by the IDR despite their diverse domain structures, suggesting the functional conservation of these domains among plant species.This study highlights the role of MEL2 granule dynamics and its impact on meiotic transition and progression. [ABSTRACT FROM AUTHOR]

A recent study published in Acta Neuropathologica identified a novel missense mutation of the small muscle protein X-linked (SMPX) gene in a Chinese pedigree. The affected individuals in the pedigree exhibited symptoms of scapuloperoneal myopathy and myofibrillar myopathy, with both proximal and distal muscle weakness and mild sensorineural deafness. Muscle biopsies revealed characteristic pathological features, including atrophic round fibers, rimmed vacuoles, and eosinophilic substances. This study expands our understanding of SMPX-related myopathy and highlights the need for further research to establish a relationship between the mutation and deafness. [Extracted from the article]

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1–244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes. SICKLE sequestrates DBR1 into stress granules to promote the accumulation of lariRNAs, which upregulate heat stress-related genes and enhance heat-stress tolerance. [ABSTRACT FROM AUTHOR]

Bag3 (Bcl-2-associated athanogene 3) protein contains a number of functional domains and interacts with a wide range of different partner proteins, including small heat shock proteins (sHsps) and heat shock protein Hsp70. The ternary Bag3–sHsp–and Hsp70 complex binds denatured proteins and transports them to phagosomes, thus playing a key role in the chaperone-assisted selective autophagy (CASA). This complex also participates in the control of formation and disassembly of stress granules (granulostasis) and cytoskeleton regulation. As Bag3 and sHsps participate in multiple cellular processes, mutations in these proteins are often associated with neurodegenerative diseases and cardiomyopathy. The review discusses the role of sHsps in different processes regulated by Bag3. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways. [ABSTRACT FROM AUTHOR]

Cellular stress response pathways (CSRPs) constantly monitor a changing microenvironment. Intrinsic and extrinsic stresses activate specific CSRPs that converge at the level of RNA metabolism, aiding cell survival. RNA G-quadruplexes (rG4s) are emerging stress-responsive cis -elements found in both the coding and noncoding transcriptomes. The dynamics of rG4s contribute to different aspects of RNA metabolism from transcription to decay. Cellular stress affects rG4s' dynamics and the functions of rG4-bearing transcripts. The rG4 proteome is highly enriched with RNA-binding proteins (RBPs) that contribute to cellular stress responses. Multiple RBPs bind rG4 transcripts and modulate their localization, stability, and translation in a stress-responsive manner. Interactions between rG4 transcripts and RBPs contribute to liquid–liquid phase separation (LLPS), the phenomenon that underlies the formation of membraneless compartments. Specifically, rG4s are implicated in the formation and dynamics of stress-responsive RNA granules, such as stress granules. Stress-induced rG4s are potential therapeutic targets for human disease. RNA G-quadruplexes (rG4s) are noncanonical secondary structures formed by guanine-rich sequences that are found in different regions of RNA molecules. These structures have been implicated in diverse biological processes, including translation, splicing, and RNA stability. Recent studies have suggested that rG4s play a role in the cellular response to stress. This review summarizes the current knowledge on rG4s under stress, focusing on their formation, regulation, and potential functions in stress response pathways. We discuss the molecular mechanisms that regulate the formation of rG4 under different stress conditions and the impact of these structures on RNA metabolism, gene expression, and cell survival. Finally, we highlight the potential therapeutic implications of targeting rG4s for the treatment of stress-related diseases through modulating cell survival. [ABSTRACT FROM AUTHOR]

Aim: Post‐transcriptional modifications and their specific mechanisms are the focus of research on the regulation of myocardial damage. Stress granules (SGs) can inhibit the inflammatory response by inhibiting the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) pathway. This study investigated whether alkylation repair homologue protein 5 (ALKBH5) could affect myocardial inflammation and apoptosis during diabetic myocardial ischaemia‐reperfusion injury (IRI) through the cGAS‐STING pathway via SGs. Methods: A diabetes ischaemia‐reperfusion rat model and a high glucose hypoxia/reoxygenation cell model were established. Adeno‐associated virus (AAV) and lentivirus (LV) were used to overexpress ALKBH5, while the SG agonist arsenite (Ars) and the SG inhibitor anisomycin were used as interventions. Then, the levels of apoptosis and related indicators in the cell and rat models were measured. Results: In the in vivo experiment, compared with the normal sham group, the degree of myocardial tissue damage, creatine kinase‐MB and cardiac troponin I in serum, and myocardial apoptosis, the infarcted area of myocardium, and the level of B‐cell lymphoma 2 associated X protein, cGAS‐STING pathway and inflammatory factors in the diabetes ischaemia‐reperfusion group were significantly increased. However, the expression of SGs and the levels of ALKBH5, rat sarcoma‐GTPase‐activating protein‐binding protein 1, T‐cell intracellular antigen‐1 and Bcl2 were significantly decreased. After AAV‐ALKBH5 intervention, the degree of myocardial tissue damage, degree of myocardial apoptosis, and extent of myocardial infarction in myocardial tissue were significantly decreased. In the in vitro experiment, compared with those in the normal control group, the levels of lactate dehydrogenase, inflammation and apoptosis were significantly greater, and cell viability and the levels of ALKBH5 and SGs were decreased in the high glucose and hypoxia/reoxygenation groups. In the high glucose hypoxia/reoxygenation cell model, the degree of cell damage, inflammation, and apoptosis was greater than those in the high glucose and hypoxia/reoxygenation models, and the levels of ALKBH5 and SGs were further decreased. LV‐ALKBH5 and Ars alleviated the degree of cell damage and inhibited inflammation and cell apoptosis. The inhibition of SGs could partly reverse the protective effect of LV‐ALKBH5. The cGAS agonist G140 antagonized the inhibitory effects of the SG agonist Ars on cardiomyocyte apoptosis, inflammation and the cGAS‐STING pathway. Conclusion: Both ALKBH5 and SGs inhibited myocardial inflammation and apoptosis during diabetic myocardial ischaemia‐reperfusion. Mechanistically, ALKBH5 might inhibit the apoptosis of cardiomyocytes by promoting the expression of SGs through the cGAS‐STING pathway. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are highly dynamic micromolecular membraneless condensates that generate in cells subjected to stress. Formed from pools of untranslating messenger ribonucleoproteins (RNP), SGs dynamics constitute vital processes essential for cell survival. Here, we investigate whether established cytotoxic agents, such as the platinum-based chemotherapeutic agent cisplatin and the aminoglycoside antibiotic gentamicin, elicit SG formation in the House Ear Institute-Organ of Corti-1 (HEI-OC1) auditory cell line, H4 human neuroglioma cells and HEK-293T human embryonic kidney cells. Cells were treated with cisplatin or gentamicin for specific durations at designated concentrations. SG formation was assessed using immunocytochemistry and live cell imaging. Levels of essential proteins involved in SG assembly were evaluated using immunoblotting. We observed cisplatin-associated SG assembly in HEI-OC1 and H4 cells via confocal microscopy through antibody colabeling of G3BP1 with PABP or Caprin1. While maintaining an unchanged pattern of expression of main constituent SG proteins, cisplatin-related SGs in H4 cells persisted for at least 12 h after drug removal. Cells subjected to gentamicin exposure did not exhibit SGs. Our findings offer insights into subcellular mechanisms related to cisplatin-associated cytotoxicity, highlighting the need for future studies to further investigate this stress-response mechanism.

Tauopathies are a heterogenous group of neurodegenerative disorders characterized by tau aggregation in the brain. In a subset of tauopathies, rare mutations in the MAPT gene, which encodes the tau protein, are sufficient to cause disease; however, the events downstream of MAPT mutations are poorly understood. Here, we investigate the role of long non-coding RNAs (lncRNAs), transcripts >200 nucleotides with low/no coding potential that regulate transcription and translation, and their role in tauopathy. Using stem cell derived neurons from patients carrying a MAPT p.P301L, IVS10 + 16, or p.R406W mutation and CRISPR-corrected isogenic controls, we identified transcriptomic changes that occur as a function of the MAPT mutant allele. We identified 15 lncRNAs that were commonly differentially expressed across the three MAPT mutations. The commonly differentially expressed lncRNAs interact with RNA-binding proteins that regulate stress granule formation. Among these lncRNAs, SNHG8 was significantly reduced in a mouse model of tauopathy and in FTLD-tau, progressive supranuclear palsy, and Alzheimer's disease brains. We show that SNHG8 interacts with tau and stress granule-associated RNA-binding protein TIA1. Overexpression of mutant tau in vitro is sufficient to reduce SNHG8 expression and induce stress granule formation. Rescuing SNHG8 expression leads to reduced stress granule formation and reduced TIA1 levels in immortalized cells and in MAPT mutant neurons, suggesting that dysregulation of this non-coding RNA is a causal factor driving stress granule formation via TIA1 in tauopathies.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed ActuAtor that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Exposure to toxic molecules from food or oral medications induces toxicity in colon cells that cause various human diseases; however, in vitro monitoring systems for colon cell toxicity are not well established. Stress granules are nonmembranous foci that form in cells exposed to cellular stress. When cells sense toxic environments, they acutely and systemically promote stress granule formation, with Ras GTPase-activating protein-binding protein 1 (G3BP1) acting as a core component to protect their mRNA from abnormal degradation. Here, we knocked in green fluorescent protein (GFP)-coding sequences into the C-terminal region of the G3BP1 gene in a human colon cell line through CRISPR-Cas9-mediated homologous recombination and confirmed the formation of stress granules with the G3BP1-GFP protein in these cells under cellular stress exposure. We demonstrated the formation and dissociation of stress granules in G3BP1-GFP expressing colon cells through real-time monitoring using a fluorescence microscope. Furthermore, we validated the toxicity monitoring system in the established colon cell line by observing stress granule formation following exposure to dihydrocapsaicin, bisphenol A, and sorbitol. Taken together, we established a stress granule reporter system in a colon cell line, providing a novel assessment for the real-time monitoring of colon toxicity in response to various chemicals.

ABSTRACTPost-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs – like TIAR and HUR – in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.

Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways.

Various stress conditions, such as heat stress (HS) and oxidative stress, can cause biomolecular condensates represented by stress granules (SGs) via liquid-liquid phase separation. We have previously shown that Hsp90 forms aggregates in response to HS and that Hsp90 aggregates transiently co-localize with SGs as visualized by Pabp. Here, we showed that arsenite, one of the well-described SG-inducing stimuli, induces Hsp90 aggregates distinct from conventional SGs in fission yeast. Arsenite induced Hsp90 granules in a dose-dependent manner, and these granules were significantly diminished by the co-treatment with a ROS scavenger N-acetyl cysteine (NAC), indicating that ROS are required for the formation of Hsp90 granules upon arsenite stress. Notably, Hsp90 granules induced by arsenite do not overlap with conventional SGs as represented by eIF4G or Pabp, while HS-induced Hsp90 granules co-localize with SGs. Nrd1, an RNA-binding protein known as a HS-induced SG component, was recruited into Hsp90 aggregates but not to the conventional SGs upon arsenite stress. The non-phosphorylatable eIF2α mutants significantly delayed the Hsp90 granule formation upon arsenite treatment. Importantly, inhibition of Hsp90 by geldanamycin impaired the Hsp90 granule formation and reduced the arsenite tolerance. Collectively, arsenite stimulates two types of distinct aggregates, namely conventional SGs and a novel type of aggregates containing Hsp90 and Nrd1, wherein Hsp90 plays a role as a center for aggregation, and stress-specific compartmentalization of biomolecular condensates.

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates. [ABSTRACT FROM AUTHOR]

Amyloid plaques are a major pathological hallmark involved in Alzheimer's disease and consist of deposits of the amyloid‐β peptide (Aβ). The aggregation process of Aβ is highly complex, which leads to polymorphous aggregates with different structures. In addition to aberrant aggregation, Aβ oligomers can undergo liquid‐liquid phase separation (LLPS) and form dynamic condensates. It has been hypothesized that these amyloid liquid droplets affect and modulate amyloid fibril formation. In this review, we briefly introduce the relationship between stress granules and amyloid protein aggregation that is associated with neurodegenerative diseases. Then we highlight the regulatory role of LLPS in Aβ aggregation and discuss the potential relationship between Aβ phase transition and aggregation. Furthermore, we summarize the current structures of Aβ oligomers and amyloid fibrils, which have been determined using nuclear magnetic resonance (NMR) and cryo‐electron microscopy (cryo‐EM). The structural variations of Aβ aggregates provide an explanation for the different levels of toxicity, shed light on the aggregation mechanism and may pave the way towards structure‐based drug design for both clinical diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Background: Methyltransferase-like 3 (METTL3), a component of the N6-methyladenosine (m6A) methyltransferase family, exhibits significant expression in HEI-OC1 cells and cochlear explants. Aminoglycoside antibiotics, known for their ototoxic potential, frequently induce irreversible auditory damage in hair cells, predominantly through oxidative stress mechanisms. However, the specific role of METTL3 in kanamycin-induced hair cell loss remains unclear. Objective: This study aims to elucidate the mechanisms by which METTL3 contributes to kanamycin-induced ototoxicity. Methods and Results: In vivo experiments demonstrated a notable reduction in METTL3 expression within cochlear explants following kanamycin administration, concomitant with the formation of stress granules (SGs). Similarly, a 24-hour kanamycin treatment led to decreased METTL3 expression and induced SG formation both in HEI-OC1 cells and neonatal cochlear explants, corroborating the in vivo observations. Lentivirus-mediated transfection was employed to overexpress and knockdown METTL3 in HEI-OC1 cells. Knockdown of METTL3 resulted in increased reactive oxygen species (ROS) levels and apoptosis induced by kanamycin, while concurrently reducing SG formation. Conversely, overexpression of METTL3 attenuated ROS generation, decreased apoptosis rates, and promoted SG formation induced by kanamycin. Therefore, METTL3-mediated SG formation presents a promising target for mitigating kanamycin-induced ROS generation and the rate of apoptosis. Conclusion: This finding indicates that METTL3-mediated SG formation holds potential in mitigating kanamycin-induced impairments in cochlear hair cells by reducing ROS formation and apoptosis rates. [ABSTRACT FROM AUTHOR]

Ribonucleoprotein (RNP) granules are membraneless electron-dense structures rich in RNAs and proteins, and involved in various cellular processes. Two RNP granules in male germ cells, intermitochondrial cement and the chromatoid body (CB), are associated with PIWI-interacting RNAs (piRNAs) and are required for transposon silencing and spermatogenesis. Other RNP granules in male germ cells, the reticulated body and CB remnants, are also essential for spermiogenesis. In this study, we disrupted FBXO24, a testis-enriched F-box protein, in mice and found numerous membraneless electron-dense granules accumulated in sperm flagella. Fbxo24 knockout (KO) mice exhibited malformed flagellar structures, impaired sperm motility, and male infertility, likely due to the accumulation of abnormal granules. The amount and localization of known RNP granule-related proteins were not disrupted in Fbxo24 KO mice, suggesting that the accumulated granules were distinct from known RNP granules. Further studies revealed that RNAs and two importins, IPO5 and KPNB1, abnormally accumulated in Fbxo24 KO spermatozoa and that FBXO24 could ubiquitinate IPO5. In addition, IPO5 and KPNB1 were recruited to stress granules, RNP complexes, when cells were treated with oxidative stress or a proteasome inhibitor. These results suggest that FBXO24 is involved in the degradation of IPO5, disruption of which may lead to the accumulation of abnormal RNP granules in sperm flagella. [ABSTRACT FROM AUTHOR]

Dysfunctional RNA‐binding proteins (RBPs) have been implicated in several geriatric diseases, including Alzheimer's disease (AD). However, little is known about the nuclear molecular actions and cooperative functions mediated by RBPs that affect gene regulation in sporadic AD or aging. In the present study, we investigated aging‐ and AD‐associated changes in the expression of PSF and G3BP2, which are representative RBPs associated with sex hormone activity. We determined that both PSF and G3BP2 levels were decreased in aged brains compared to young brains of mice. RNA sequencing (RNA‐seq) analysis of human neuronal cells has shown that PSF is responsible for neuron‐specific functions and sustains cell viability. In addition, we showed that PSF interacted with G3BP2 in the nucleus and stress granules (SGs) at the protein level. Moreover, PSF–mediated gene regulation at the RNA level correlated with G3BP2. Interestingly, PSF and G3BP2 target genes are associated with AD development. Mechanistically, quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis demonstrated that the interaction of RBPs with the pre‐mRNA of target genes enhanced post‐transcriptional mRNA stability, suggesting a possible role for these RBPs in preserving neuronal cell viability. Notably, in the brains of patients with sporadic AD, decreased expression of PSF and G3BP2 in neurons was observed compared to non‐AD patients. Overall, our findings suggest that the cooperative action of PSF and G3BP2 in the nucleus is important for preventing aging and AD development. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid–liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated. [ABSTRACT FROM AUTHOR]

Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras- GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate. [ABSTRACT FROM AUTHOR]

Neurodevelopmental disorders (NDDs) represent a large group of disorders with an onset in the neonatal or early childhood period; NDDs include intellectual disability (ID), autism spectrum disorders (ASD), attention deficit hyperactivity disorders (ADHD), seizures, various motor disabilities and abnormal muscle tone. Among the many underlying Mendelian genetic causes for these conditions, genes coding for proteins involved in all aspects of the gene expression pathway, ranging from transcription, splicing, translation to the eventual RNA decay, feature rather prominently. Here we focus on two large families of RNA helicases (DEAD- and DExH-box helicases). Genetic variants in the coding genes for several helicases have recently been shown to be associated with NDD. We address genetic constraints for helicases, types of pathological variants which have been discovered and discuss the biological pathways in which the affected helicase proteins are involved. [ABSTRACT FROM AUTHOR]

Background: The global outbreak of COVID-19 caused by the SARS-CoV-2 has led to millions of deaths. This unanticipated emergency has prompted virologists across the globe to delve deeper into the intricate dynamicity of the host-virus interface with an aim to identify antiviral targets and elucidate host and viral determinants of severe disease. Aim: The present study was undertaken to analyse the role of histone deacetylase 6 (HDAC6) in regulating SARS-CoV-2 infection. Results: Gradual increase in HDAC6 expression was observed in different SARS-CoV-2-permissive cell lines following SARS-CoV-2 infection. The SARS-CoV-2 nucleocapsid protein (N protein) was identified as the primary viral factor responsible for upregulating HDAC6 expression. Downregulation of HDAC6 using shRNA or a specific inhibitor tubacin resulted in reduced viral replication suggesting proviral role of its deacetylase activity. Further investigations uncovered the interaction of HDAC6 with stress granule protein G3BP1 and N protein during infection. HDAC6-mediated deacetylation of SARS-CoV-2 N protein was found to be crucial for its association with G3BP1. Conclusion: This study provides valuable insights into the molecular mechanisms underlying the disruption of cytoplasmic stress granules during SARS-CoV-2 infection and highlights the significance of HDAC6 in the process. [ABSTRACT FROM AUTHOR]

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD. [ABSTRACT FROM AUTHOR]

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress. [ABSTRACT FROM AUTHOR]

Although the cause of interstitial cystitis/painful bladder syndrome (IC/PBS) remains unknown, autoimmune involvement has been strongly suggested to be a contributing factor. To elucidate the pathophysiology of IC/PBS, we characterized the experimental autoimmune cystitis (EAC) in rats. Adult female Sprague-Dawley rats were divided into the EAC and control groups. The EAC rats were generated by administrating a homogenate of donor rat bladder tissue as a bladder antigen. The characteristics of the two groups were determined by evaluating pain behavior and conducting cystometry, histopathology, and molecular analyses. The EAC rats showed: 1) a decreased paw withdrawal threshold, 2) a reduced intercontraction interval on cystometry, 3) the irregular surfaces of the umbrella cells of epithelium throughout the bladder wall, 4) accumulation of stress granules in the bladder and vascular endothelium, 5)the increased expression of genes related to inflammation and ischemia at the mRNA and protein levels, 6) a significantly increased paw withdrawal threshold with pain treatment, and 7) the induction of glomerulation of the bladder wall, epithelium denudation, and lymphocyte infiltration in the interstitium by bladder distension. These results suggest that the EAC rats showed pain and frequent urination with the overexpression of inflammatory chemokines, reflecting clinical IC/BPS, and the bladder epithelium and vascular endothelium may be the primary sites of IC/BPS, and bladder injury, such as bladder distension, can cause progression from BPS to IC with Hunner lesions. NEW & NOTEWORTHY: The experimental autoimmune cystitis model rats showed pain and frequent urination with the overexpression of inflammatory chemokines, reflecting clinical interstitial cystitis/painful bladder syndrome (IC/PBS), and the bladder epithelium and vascular endothelium may be the primary sites of IC/BPS, and bladder injury, such as bladder distension, can cause progression from BPS to IC with Hunner lesions. [ABSTRACT FROM AUTHOR]

Pathogenic variants in hnRNPA1 have been reported in amyotrophic lateral sclerosis (ALS) patients. However, studies on hnRNPA1 mutant spectrum and pathogenicity of variants were rare. We performed whole exome sequencing of ALS-associated genes and subsequent verification of rare variants in hnRNPA1 in our ALS patients. The hnRNPA1 mutations reported in literature were reviewed and combined with our results to determine the genotype-phenotype relationship. Functional analysis of the novel variant p.G195A was performed in vitro by transfection of mutant hnRNPA1 into 293T cell. Among 207 ALS patients recruited, 3 rare hnRNPA1 variants were identified (mutant frequency 1.45%), including two recurrent mutations (p.P340S and p.G283R), and a novel rare variant p.G195A. In combination with previous reports, there are 27 ALS patients with 15 hnRNPA1 mutations identified. Disease onset age was 47.90 ± 1.52 years with predominant limb onset. The p.P340S mutation caused flail arm syndrome (FAS) in two independent families with extended life expectancy. The newly identified p.G195A mutation, lying at the start of the PrLD ("prion-like" domain)/LCD (low-complexity domain), causes local structural changes in 3D protein prediction. Upon sodium arsenite exposure, mutant hnRNPA1 retained in the nucleus but deficit of cytoplasmic G3BP1-positive stress granule clearance was observed. This is different from the p.P340S mutation which caused both cytoplasmic translocation and stress granule formation. No cytoplasmic TDP-43 translocation was observed. Mutations in hnRNPA1 are overall minor in ALS patients. The p.P340S mutation is associated with manifestation of FAS. Mutations in LCD of hnRNPA1 cause stress granule misprocessing. [ABSTRACT FROM AUTHOR]

SUMMARY: Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein–RNA, protein–DNA, and protein–protein interactions. Here, we reveal an integrated multi‐level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15‐inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING‐type E3 ligase MED25‐BINDING RING‐H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild‐type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2‐dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss‐of‐function and decreased in MBR2 overexpression backgrounds. Heat stress‐induced accumulation of HSC70.3 promoted MBR2‐mediated degradation of C3H15 protein, implying that an auto‐regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops. Significance Statement: Heat stress is a severe environmental factor that significantly inhibits plant growth and development, but the molecular mechanisms governing plant responses to heat stress are not fully understood. This study reveals a mechanism of multi‐level regulation for thermotolerance in Arabidopsis, which will facilitate creating heat‐tolerant crops. [ABSTRACT FROM AUTHOR]

Plants, as sessile organisms, face the crucial challenge of adjusting growth and development with ever-changing environmental conditions. Protein synthesis is the fundamental process that enables growth of all organisms. Since elevated temperature presents a substantial threat to protein stability and function, immediate adjustments of protein synthesis rates are necessary to circumvent accumulation of proteotoxic stress and to ensure survival. This review provides an overview of the mechanisms that control translation under high-temperature stress by the modification of components of the translation machinery in plants, and compares them to yeast and metazoa. Recent research also suggests an important role for cytoplasmic biomolecular condensates, named stress granules, in these processes. Current understanding of the role of stress granules in translational regulation and of the molecular processes associated with translation that might occur within stress granules is also discussed. [ABSTRACT FROM AUTHOR]