The c-Myc oncoprotein plays a prominent role in cancer initiation, progression, and maintenance. Long noncoding RNAs (lncRNAs) are recently emerging as critical regulators of the c-Myc signaling pathway. Here, we report the lncRNA USP2-AS1 as a direct transcriptional target of c-Myc. Functionally, USP2-AS1 inhibits cellular senescence and acts as an oncogenic molecule by inducing E2F1 expression. Mechanistically, USP2-AS1 associates with the RNA-binding protein G3BP1 and facilitates the interaction of G3BP1 to E2F1 3'-untranslated region, thereby leading to the stabilization of E2F1 messenger RNA. Furthermore, USP2-AS1 is shown as a mediator of the oncogenic function of c-Myc via the regulation of E2F1. Together, these findings suggest that USP2-AS1 is a negative regulator of cellular senescence and also implicates USP2-AS1 as an important player in mediating c-Myc function.

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

A recent study by Gwon et al. identified context-specific ubiquitination of G3BP1 as critical for stress granule disassembly via VCP and the adaptor FAF2. This study provides new insights into stress granule dynamics, with potential implications for neurodegenerative disease.

Keywords: G3BP1; RNA granules; amyotrophic lateral sclerosis; frontotemporal dementia; neurodegeneration; pathomechanism.

SPOP, an E3 ubiquitin ligase, acts as a prostate-specific tumor suppressor with several key substrates mediating oncogenic function. However, the mechanisms underlying SPOP regulation are largely unknown. Here, we have identified G3BP1 as an interactor of SPOP and functions as a competitive inhibitor of Cul3^SPOP, suggesting a distinctive mode of Cul3^SPOP inactivation in prostate cancer (PCa). Transcriptomic analysis and functional studies reveal a G3BP1-SPOP ubiquitin signaling axis that promotes PCa progression through activating AR signaling. Moreover, AR directly upregulates G3BP1 transcription to further amplify G3BP1-SPOP signaling in a feed-forward manner. Our study supports a fundamental role of G3BP1 in disabling the tumor suppressive Cul3^SPOP, thus defining a PCa cohort independent of SPOP mutation. Therefore, there are significantly more PCa that are defective for SPOP ubiquitin ligase than previously appreciated, and these G3BP1^high PCa are more susceptible to AR-targeted therapy.

[This corrects the article DOI: 10.3389/fimmu.2018.01142.].

Keywords: G3BP stress granule assembly factor 1; foot-and-mouth disease virus; innate immunity; internal ribosome entry site; phosphoproteomics.

Background: Small cell lung cancer (SCLC) is lethal and possesses limited therapeutic options. Platinum-based chemotherapy-with or without immune checkpoint inhibitors (anti-PDs)-is the current first-line therapy for SCLCs; however, its associated outcomes are heterogeneous. N⁶-methyladenosine (m⁶A) is a novel and decisive factor in tumour progression, chemotherapy resistance, and immunotherapy response. However, m⁶A modification in SCLC remains poorly understood.

Methods: We systematically explored the molecular features and clinical significance of m⁶A regulators in SCLC. We then constructed an m⁶A regulator-based prognostic signature (m⁶A score) based on our examination of 256 cases with limited-stage SCLC (LS-SCLC) from three different cohorts-including an independent cohort that contained 150 cases with qPCR data. We additionally evaluated the relationships between the m⁶A score and adjuvant chemotherapy (ACT) benefits and the patients' responses to anti-PD-1 treatment. Immunohistochemical (IHC) staining and the HALO digital pathological platform were used to calculate CD8+ T cell density.

Results: We observed abnormal somatic mutations and expressions of m⁶A regulators. Using the LASSO Cox model, a five-regulator-based (G3BP1, METTL5, ALKBH5, IGF2BP3, and RBM15B) m⁶A score was generated from the significant regulators to classify patients into high- and low-score groups. In the training cohort, patients with high scores had shorter overall survival (HR, 5.19; 2.75-9.77; P < 0.001). The prognostic accuracy of the m⁶A score was well validated in two independent cohorts (HR 4.6, P = 0.006 and HR 3.07, P < 0.001). Time-dependent ROC and C-index analyses found the m⁶A score to possess superior predictive power than other clinicopathological parameters. A multicentre multivariate analysis revealed the m⁶A score to be an independent prognostic indicator. Additionally, patients with low scores received a greater survival benefit from ACT, exhibited more CD8+ T cell infiltration, and were more responsive to cancer immunotherapy.

Conclusions: Our results, for the first time, affirm the significance of m⁶A regulators in LS-SCLC. Our multicentre analysis found that the m⁶A score was a reliable prognostic tool for guiding chemotherapy and immunotherapy selections for patients with SCLC.

Keywords: Chemotherapy; Immunotherapy; Individualized medicine; Small cell lung cancer; m6A regulators.

Background: Circular RNAs (circRNAs) represent a novel class of non-coding RNAs formed by a covalently closed loop and play crucial roles in many biological processes. Several circRNAs associated with myogenesis have been reported. However, the dynamic expression, function, and mechanism of circRNAs during myogenesis and skeletal muscle development are largely unknown.

Methods: Strand-specific RNA-sequencing (RNA-seq) and microarray datasets were used to profile the dynamic circRNAome landscape during skeletal muscle development and myogenic differentiation. Bioinformatics analyses were used to characterize the circRNAome and identify candidate circRNAs associated with myogenesis. Bulk and single-cell RNA-seq were performed to identify the downstream genes and pathways of circFgfr2. The primary myoblast cells, C2C12 cells, and animal model were used to assess the function and mechanism of circFgfr2 in myogenesis and muscle regeneration in vitro or in vivo by RT-qPCR, western blotting, dual-luciferase activity assay, RNA immunoprecipitation, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation.

Results: We profiled the dynamic circRNAome in pig skeletal muscle across 27 developmental stages and detected 52 918 high-confidence circRNAs. A total of 2916 of these circRNAs are conserved across human, mouse, and pig, including four circRNAs (circFgfr2, circQrich1, circMettl9, and circCamta1) that were differentially expressed (|log₂ fold change| > 1 and adjusted P value < 0.05) in various myogenesis systems. We further focused on a conserved circRNA produced from the fibroblast growth factor receptor 2 (Fgfr2) gene, termed circFgfr2, which was found to inhibit myoblast proliferation and promote differentiation and skeletal muscle regeneration. Mechanistically, circFgfr2 acted as a sponge for miR-133 to regulate the mitogen-activated protein kinase kinase kinase 20 (Map3k20) gene and JNK/MAPK pathway. Importantly, transcription factor Kruppel like factor 4 (Klf4), the downstream target of the JNK/MAPK pathway, directly bound to the promoter of circFgfr2 and affected its expression via an miR-133/Map3k20/JNK/Klf4 auto-regulatory feedback loop. RNA binding protein G3BP stress granule assembly factor 1 (G3bp1) inhibited the biogenesis of circFgfr2.

Conclusions: The present study provides a comprehensive circRNA resource for skeletal muscle study. The functional and mechanistic analysis of circFgfr2 uncovered a circRNA-mediated auto-regulatory feedback loop regulating myogenesis and muscle regeneration, which provides new insight to further understand the regulatory mechanism of circRNAs.

Keywords: Development; Feedback loop; Regeneration; Skeletal muscle; circFgfr2; circRNA.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential innate immune sensor. Remarkably, in addition to its role in the early detection of pathogenic DNA molecules, cGAS also monitors cellular health through the sensing of nuclear and mitochondrial DNA aberrantly localised to the cell cytoplasm. This central position of cGAS requires tight molecular controls which are only starting to be understood. In this issue of EMBO Reports, Zhao and colleagues (Zhao et al, 2021) describe a novel mechanism switching on DNA sensing, relying on the formation of primary condensates of cGAS and GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1).

Stress granules (SGs) are non-membrane ribonucleoprotein condensates formed in response to environmental stress conditions via liquid-liquid phase separation (LLPS). SGs are involved in the pathogenesis of aging and aging-associated diseases, cancers, viral infection, and several other diseases. GTPase-activating protein (SH3 domain)-binding protein 1 and 2 (G3BP1/2) is a key component and commonly used marker of SGs. Recent studies have shown that SARS-CoV-2 nucleocapsid protein via sequestration of G3BPs inhibits SGs formation in the host cells. In this study, we have identified putative miRNAs targeting G3BP in search of modulators of the G3BP expression. These miRNAs could be considered as new therapeutic targets against COVID-19 infection via the regulation of SG assembly and dynamics.

Keywords: COVID-19; G3BP; Nucleocapsid; SARS-CoV-2; Stress granule; microRNA.

The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3'UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3'UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.

Keywords: Decitabine; G3BP1/2; Gene set enrichment; Imatinib; Nucleocapsid protein; SARS-CoV-2; Stress granule.

G3BP1 exists as a helicase and one of the core components in stress granules, which are associated with a variety of neurodegenerative diseases. Its RNA recognition motif (RRM) domain performs the paramount function of binding mRNA. Here we report the resonance assignment of human G3BP1 RRM domain to understand its structure-function relationship.

Keywords: G3BP1; RRM domain; Resonance assignment; Stress granule.

Stress granules (SGs) are cytosolic, nonmembranous RNA-protein (RNP) complexes that form in the cytosol of many cells under various stress conditions and can integrate responses to various stressors. Although physiological SG formation appears to be an adaptive and survival-promoting mechanism, inappropriate formation or chronic persistence of SGs has been linked to aging and various neurodegenerative diseases. The quantitative monitoring of the dynamics of SG components in living nerve cells can therefore be an important tool for identifying conditions that disrupt SG function and lead to disease-related attacks in the cells. Here, we describe a method for the quantitative determination of the distribution and shuttling dynamics of components of SGs in living model neurons by fluorescence decay after photoactivation (FDAP) measurements using a standard confocal laser scanning microscope. The method includes lipofection of photoactivatable green fluorescent protein (paGFP) fused to an SG protein of interest in a neural cell line, differentiation of the cells into a neuronal phenotype, focal activation using a blue diode (405 nm), and recording of decay curves with a 488 nm laser line. By modeling the decay measurements with FDAP functions, the approach enables estimating the residence time of the SG protein of interest, determining the proportion of the respective component in SGs, and the detection of possible changes after experimental manipulation.

Keywords: Fluorescence decay after photoactivation; G3BP1; IMP1; Liquid-liquid phase separation; Model neurons; RNA-binding proteins; Stress granules.

Long interspersed element type 1 (LINE-1, also L1 for short) is the only autonomously transposable element in the human genome. Its insertion into a new genomic site may disrupt the function of genes, potentially causing genetic diseases. Cells have thus evolved a battery of mechanisms to tightly control LINE-1 activity. Here, we report that a cellular antiviral protein, myxovirus resistance protein B (MxB), restricts the mobilization of LINE-1. This function of MxB requires the nuclear localization signal located at its N-terminus, its GTPase activity and its ability to form oligomers. We further found that MxB associates with LINE-1 protein ORF1p and promotes sequestration of ORF1p to G3BP1-containing cytoplasmic granules. Since knockdown of stress granule marker proteins G3BP1 or TIA1 abolishes MxB inhibition of LINE-1, we conclude that MxB engages stress granule components to effectively sequester LINE-1 proteins within the cytoplasmic granules, thus hindering LINE-1 from accessing the nucleus to complete retrotransposition. Thus, MxB protein provides one mechanism for cells to control the mobility of retroelements.

Ras-GTPase-activating protein binding protein 1 (G3BP1) is a multifunctional binding protein involved in a variety of biological functions, including cell proliferation, metastasis, apoptosis, differentiation and RNA metabolism. It has been revealed that G3BP1, as an antiviral factor, can interact with viral proteins and regulate the assembly of stress granules (SGs), which can inhibit viral replication. Furthermore, several viruses have the ability to hijack G3BP1 as a cofactor, recruiting translation initiation factors to promote viral proliferation. However, many functions of G3BP1 are associated with other diseases. In various cancers, G3BP1 is a cancer-promoting factor, which can promote the proliferation, invasion and metastasis of cancer cells. Moreover, compared with normal tissues, G3BP1 expression is higher in tumor tissues, indicating that it can be used as an indicator for cancer diagnosis. In this review, the structure of G3BP1 and the regulation of G3BP1 in multiple dimensions are described. In addition, the effects and potential mechanisms of G3BP1 on various carcinomas, viral infections, nervous system diseases and cardiovascular diseases are elucidated, which may provide a direction for clinical applications of G3BP1 in the future.

Keywords: Ras-GTPase-activating protein binding protein 1; carcinoma; stress granule; virus.

SARS-CoV-2 infection has caused a global pandemic that has severely damaged both public health and the economy. The nucleocapsid protein of SARS-CoV-2 is multifunctional and plays an important role in ribonucleocapsid formation and viral genome replication. In order to elucidate its functions, interaction partners of the SARS-CoV-2 N protein in human cells were identified via affinity purification and mass spectrometry. We identified 160 cellular proteins as interaction partners of the SARS-CoV-2 N protein in HEK293T and/or Calu-3 cells. Functional analysis revealed strong enrichment for ribosome biogenesis and RNA-associated processes, including ribonucleoprotein complex biogenesis, ribosomal large and small subunits biogenesis, RNA binding, catalysis, translation and transcription. Proteins related to virus defence responses, including MOV10, EIF2AK2, TRIM25, G3BP1, ZC3HAV1 and ZCCHC3 were also identified in the N protein interactome. This study comprehensively profiled the viral-host interactome of the SARS-CoV-2 N protein in human cells, and the findings provide the basis for further studies on the pathogenesis and antiviral strategies for this emerging infection.

Keywords: SARS-CoV-2; ribonucleocapsid; viral–host interactome.

Introduction: Aluminum is a kind of chemical contaminants in food which can induce neurotoxicity. Aluminum exposure is closely related to neurodegenerative diseases (ND), in which neuroinflammation might involve. However, the molecular mechanism of aluminum-induced neuroinflammation through pyroptosis is not fully clarified yet.

Material and methods: The mice model of subacute exposure to aluminum chloride (AlCl₃) was established. BV2 microglia cells was treated with AlCl₃ in vitro. Resveratrol (Rsv) was adopted as intervention agent.

Results: Our results showed that aluminum induced cognitive impairment, destroying blood brain barrier (BBB), and causing nerve injury in mice. Meanwhile, aluminum could stimulate nucleotide oligomerization domain-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome assembly and activate caspase-1 (CASP1), inducing gasdermin D (GSDMD)-mediated pyroptosis signaling, releasing cytokines IL-1β and IL-18, further promoting the activation of glial cells to magnify neuroinflammatory response. Moreover, DEAD-box helicase 3 X-linked (DDX3X) and stress granule RasGAP SH3-domain-binding protein 1 (G3BP1) both participated in neuroinflammation induced by aluminum. When co-treated with Rsv, these injuries were alleviated to some extent.

Conclusion: Aluminum exposure could induce nerve cell pyroptosis and neuroinflammation by DDX3X-NLRP3 inflammasome signaling pathway, which could be rescued via Rsv activating sirtuin 1 (SIRT1).

Keywords: Aluminum exposure; Inflammasome assembly; Neuroinflammation; Pyroptosis; Resveratrol.

RNA guanine quadruplexes (rG4) assume important roles in post-transcriptional regulations of gene expression, which are often modulated by rG4-binding proteins. Hence, understanding the biological functions of rG4s requires the identification and functional characterizations of rG4-recognition proteins. By employing a bioinformatic approach based on the analysis of overlap between peaks obtained from rG4-seq analysis and those detected in >230 eCLIP-seq datasets for RNA-binding proteins generated from the ENCODE project, we identified a large number of candidate rG4-binding proteins. We showed that one of these proteins, G3BP1, is able to bind directly to rG4 structures with high affinity and selectivity, where the binding entails its C-terminal RGG domain and is further enhanced by its RRM domain. Additionally, our seCLIP-Seq data revealed that pyridostatin, a small-molecule rG4 ligand, could displace G3BP1 from mRNA in cells, with the most pronounced effects being observed for the 3'-untranslated regions (3'-UTR) of mRNAs. Moreover, luciferase reporter assay results showed that G3BP1 positively regulates mRNA stability through its binding with rG4 structures. Together, we identified a number of candidate rG4-binding proteins and validated that G3BP1 can bind directly with rG4 structures and regulate the stabilities of mRNAs.

The assembly of stress granules (SGs) is a well-known cellular strategy for reducing stress-related damage and promoting cell survival. SGs have become important players in human health, in addition to their fundamental role in the stress response. The critical role of SGs in cancer cells in formation, progression, and metastasis makes sense. Recent researchers have found that several SG components play a role in tumorigenesis and cancer metastasis via tumor-associated signaling pathways and other mechanisms. Gene-ontology analysis revealed the role of these protein components in the structure of SGs. Involvement in the translation process, regulation of mRNA stability, and action in both the cytoplasm and nucleus are among the main features of SG proteins. The present scoping review aimed to consider all studies on the effect of SGs on cancer formation, proliferation, and metastasis and performed based on a six-stage methodology structure and the PRISMA guideline. A systematic search of seven databases for qualified articles was conducted before July 2021. Publications were screened, and quantitative and qualitative analysis was performed on the extracted data. Go analysis was performed on seventy-one SGs protein components. Remarkably G3BP1, TIA1, TIAR, and YB1 have the largest share among the proteins considered in the studies. Altogether, this scoping review tries to demonstrate and provide a comprehensive summary of the role of SGs in the formation, progression, and metastasis of cancer by reviewing all studies.

Keywords: G3BP1; TIA1; TIAR; YB1; cancer; metastasis; progression; stress granules.

When exposed to stressful conditions, eukaryotic cells respond by inducing the formation of cytoplasmic ribonucleoprotein complexes called stress granules. Here we use C. elegans to study two proteins that are important for stress granule assembly in human cells: PQN-59, the human UBAP2L ortholog, and GTBP-1, the human G3BP1/2 ortholog. Both proteins assemble into stress granules in the embryo and in the germline when C. elegans is exposed to stressful conditions. None of the two proteins is essential for the assembly of stress-induced granules, as shown by the single and combined depletions by RNAi, and neither pqn-59 nor gtbp-1 mutant embryos show higher sensitivity to stress than control embryos. We find that pqn-59 mutants display reduced progeny and a high percentage of embryonic lethality, phenotypes that are not dependent on stress exposure and that are not shared with gtbp-1 mutants. Our data indicate that, in contrast to human cells, PQN-59 and GTBP-1 are not required for stress granule formation but that PQN-59 is important for C. elegans development.

Keywords: C. elegans; GTBP-1; PQN-59; UBAP2L; development; stress granules.

Among urological tumors, renal cell carcinoma (RCC) is the third-highest mortality rate tumor, and 20%-30% of RCC patients present with metastases at the time of diagnosis. While the treatment of RCC has been improved over the last few years, its mortality stays high. Y-box binding protein 1 (YBX1) is a well-known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Our previous studies showed that YBX1 in RCC cells significantly promoted cell adhesion, migration, and invasion. However, the role of YBX1 in RCC cells apoptosis has not been reported. In this study, we investigated the effect of YBX1 on cell apoptosis and elucidated the mechanisms involved. Results showed that YBX1 regulated RCC cells apoptosis and reactive oxygen species (ROS) generation via Kindlin-2. These findings indicated that YBX1 inhibited RCC cells apoptosis and may serve as a candidate RCC prognostic marker and a potential therapeutic target. Abbreviations: RCC: Renal cell carcinoma; YBX1: Y-box binding protein 1; ROS: Reactive oxygen species; ccRCC: Clear cell renal cell carcinoma; mccRCC: Metastatic clear cell renal cell carcinoma; G3BP1: Ras-GTPase activating protein SH3 domain-binding proteins 1; SPP1: Secreted phosphoprotein 1; NF-κB: Nuclear factor kappa beta; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; PYCR1: Pyrroline-5-carboxylate reductase 1; MEM: Eagle's Minimum Essential Medium; DMEM: Dulbecco's modified Eagle medium; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; BSA: Bovine serum albumin; DCFH-DA: 2,7-Dichlorodihydrofluorescein diacetate; FITC: Fluorescein isothiocyanate; PI: Propidium iodide.

Keywords: Renal cell carcinoma; apoptosis; reactive oxygen species; s-2; y-box binding protein 1.

To rapidly adapt to stresses such as infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence and thus viruses need to evade these responses to propagate. We previously showed that Feline Calicivirus (FCV) impairs SGs assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, that we named paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.

Keywords: G3BP1; Stress granules; Viruses.

Background: Ovarian follicle fluid (FF) as a microenvironment surrounding oocyte plays critical roles in physio-biochemical processes of follicle development and oocyte maturation. It is hypothesized that proteins in yak FF participate in the physio-biochemical pathways. The primary aims of this study were to find differentially expressed proteins (DEPs) between mature and immature FF, and to elucidating functions of the mature and immature FF in yak.

Results: The mature and immature FF samples were obtained from three healthy yaks that were nonpregnant, aged from four to five years, and free from any anatomical reproductive disorders. The FF samples were subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ). The FF samples went through correlation analysis, principle component analysis, and expression pattern analysis based on quantification of the identified proteins. Four hundred sixty-three DEPs between mature and immature FF were identified. The DEPs between the mature and immature FF samples underwent gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis. The DEPs highly expressed in the mature FF mainly took parts in the complement and coagulation cascades, defense response, acute-phase response, response to other organism pathways to avoid invasion of exogenous microorganisms. The complement activation pathway contains eight DEPs, namely C2, C5, C6, C7, C9, C4BPA, CFH, and MBL2. The three DEPs, CATHL4, CHGA, and PGLYRP1, take parts in defense response pathway to prevent invasion of exogenetic microorganism. The coagulation cascades pathway involves many coagulation factors, such as F7, F13A1, FGA, FGB, FGG, KLKB1, KNG1, MASP1, SERPINA1, and SERPIND1. While the DEPs highly expressed in the immature FF participated in protein translation, peptide biosynthetic process, DNA conformation change, and DNA geometric change pathways to facilitate follicle development. The translation pathway contains many ribosomal proteins, such as RPL3, RPL5, RPS3, RPS6, and other translation factors, such as EIF3J, EIF4G2, ETF1, MOV10, and NARS. The DNA conformation change and DNA geometric change involve nine DEPs, DDX1, G3BP1, HMGB1, HMGB2, HMGB3, MCM3, MCM5, MCM6, and RUVBL2. Furthermore, the expressed levels of the main DEPs, C2 and SERPIND1, were confirmed by western blot.

Conclusions: The differential proteomics revealed the up-regulated DEPs in mature FF take parts in immunoreaction to prevent invasion of microorganisms and the up-regulated DEPs in immature FF participate in protein synthesis, which may improve our knowledge of the follicular microenvironment and its biological roles for reproductive processes in yak. The DEPs, C2 and SERPIND1, can be considered as protein markers for mature yak follicle.

Keywords: Immune reaction; Ovarian follicle; Protein translation; Proteomics; Yak; iTRAQ.