scholarly journals The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation

2010 ◽  
Vol 427 (3) ◽  
pp. 349-357 ◽  
Author(s):  
Shantá D. Hinton ◽  
Michael P. Myers ◽  
Vincent R. Roggero ◽  
Lizabeth A. Allison ◽  
Nicholas K. Tonks
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Active Form ◽  
Protein Tyrosine Phosphatases ◽  
Stress Granules ◽  
Stress Granule ◽  
Mitogen Activated Protein Kinase ◽  
Granule Formation ◽  
Dual Specificity ◽  
Catalytically Active

MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to ‘stress granules’ after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.

scholarly journals Regulation of Stress Granule Formation by Inflammation, Vascular Injury, and Atherosclerosis

2019 ◽  
Vol 39 (10) ◽  
pp. 2014-2027 ◽  
Author(s):  
Allison B. Herman ◽  
Milessa Silva Afonso ◽  
Sheri E. Kelemen ◽  
Mitali Ray ◽  
Christine N. Vrakas ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Rna Binding ◽  
Binding Protein ◽  
Muscle Cells ◽  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Granule Formation ◽  
Altered Expression

Objective: Stress granules (SGs) are dynamic cytoplasmic aggregates containing mRNA, RNA-binding proteins, and translation factors that form in response to cellular stress. SGs have been shown to contribute to the pathogenesis of several human diseases, but their role in vascular diseases is unknown. This study shows that SGs accumulate in vascular smooth muscle cells (VSMCs) and macrophages during atherosclerosis. Approach and Results: Immunohistochemical analysis of atherosclerotic plaques from LDLR − /− mice revealed an increase in the stress granule-specific markers Ras-G3BP1 (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP1+ SGs were rapidly induced in VSMC and bone marrow–derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α (eukaryotic translation initiation factor 2-alpha) phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. Conclusions: These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.

scholarly journals The RNA binding protein Musashi1 regulates apoptosis, gene expression and stress granule formation in urothelial carcinoma cells

2010 ◽  
Vol 15 (5) ◽  
pp. 1210-1224 ◽  
Author(s):  
Parvaneh Nikpour ◽  
Modjtaba Emadi Baygi ◽  
Christine Steinhoff ◽  
Christiane Hader ◽  
Anna C. Luca ◽  
...  
Keyword(s):  
Gene Expression ◽  
Urothelial Carcinoma ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Carcinoma Cells ◽  
Stress Granule ◽  
Granule Formation ◽  
Apoptosis Gene

scholarly journals Chicken RNA-binding protein T-cell internal antigen-1 contributes to stress granule formation in chicken cells and tissues

2018 ◽  
Vol 19 (1) ◽  
pp. 3
Author(s):  
Yingjie Sun ◽  
Pin Zhang ◽  
Hang Zheng ◽  
Luna Dong ◽  
Lei Tan ◽  
...  
Keyword(s):  
T Cell ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stress Granule ◽  
Granule Formation

scholarly journals U2AF1 Mutations Enhance Stress Granule Response in Myeloid Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 321-321
Author(s):  
Giulia Biancon ◽  
Poorval Joshi ◽  
Joshua T Zimmer ◽  
Torben Hunck ◽  
Yimeng Gao ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Binding ◽  
Stress Granules ◽  
Stress Granule ◽  
Confocal Imaging ◽  
Splicing Factor ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Wild Type ◽  
Granule Formation

Abstract Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.

scholarly journals Polyadenylated RNA and RNA-Binding Proteins Exhibit Unique Response to Hyperosmotic Stress

2021 ◽  
Vol 9 ◽  
Author(s):  
Benjamin L. Zaepfel ◽  
Jeffrey D. Rothstein
Keyword(s):  
Osmotic Stress ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Localization ◽  
Stress Granules ◽  
Stress Granule ◽  
Granule Formation ◽  
Polyadenylated Rna ◽  
And Function

Stress granule formation is a complex and rapidly evolving process that significantly disrupts cellular metabolism in response to a variety of cellular stressors. Recently, it has become evident that different chemical stressors lead to the formation of compositionally distinct stress granules. However, it is unclear which proteins are required for the formation of stress granules under different conditions. In addition, the effect of various stressors on polyadenylated RNA metabolism remains enigmatic. Here, we demonstrate that G3BP1/2, which are common stress granule components, are not required for the formation of stress granules specifically during osmotic stress induced by sorbitol and related polyols. Furthermore, sorbitol-induced osmotic stress leads to significant depletion of nuclear polyadenylated RNA, a process that we demonstrate is dependent on active mRNA export, as well as cytoplasmic and subnuclear shifts in the presence of many nuclear RNA-binding proteins. We assessed the function of multiple shifted RBPs and found that hnRNP U, but not TDP-43 or hnRNP I, exhibit reduced function following this cytoplasmic shift. Finally, we observe that multiple stress pathways lead to a significant reduction in transcription, providing a possible explanation for our inability to observe loss of TDP-43 or hnRNP I function. Overall, we identify unique outcomes following osmotic stress that provide important insight into the regulation of RNA-binding protein localization and function.

scholarly journals The Orb6-Sts5 Axis Regulates Stress Granule Formation and Heat Stress Response in Fission Yeast

2021 ◽  
Author(s):  
Robert N. Tams ◽  
Chuan Chen ◽  
Illyce Nuñez ◽  
Patrick Roman Haller ◽  
Fulvia Verde
Keyword(s):  
Heat Stress ◽  
Cell Growth ◽  
Cell Survival ◽  
Rna Binding ◽  
Stress Granules ◽  
Stress Granule ◽  
Granule Formation ◽  
Heat Stress Response ◽  
P Bodies ◽  
Intrinsically Disordered

AbstractThe NDR/LATS family kinases are a subclass of the AGC serine/threonine kinases which are important for morphogenesis and cell growth control. Using the model organismSchizosaccharomyces pombe, we previously reported that the NDR/LATS kinase Orb6 phosphorylates the RNA-binding protein (RBP) Sts5 serine 86 residue on its Intrinsically Disordered Domain (IDD). When dephosphorylated, Sts5 forms ribonucleoprotein (RNP) granules that colocalize with processing bodies (P-Bodies) and translationally repress mRNAs important for polarized cell growth. Here we report that Sts5 puncta colocalize with both P-Bodies and stress granules (SG) in response to glucose starvation, as well as heat, oxidative, and hyperosmotic stress. We find that loss of Sts5 decreases the number of stress granules, indicating that Sts5 has a role in promoting stress granule formation. Conversely, inhibition of Orb6 kinase promotes Sts5 aggregation and stress granule formation. In addition, loss of Sts5 decreases cell survival after heat stress, whereas decreasing Orb6 protein levels or including thests5S86Amutation, which promotes Sts5 aggregation, leads to increased survival. These data indicate that the Orb6-Sts5 axis is not only important for regulation of polarized growth but also for response to environmental stress, as dysregulation of the Orb6-Sts5 axis affects stress granule formation and cell survival.

scholarly journals Targeted deletion of the RNA-binding protein Caprin1 leads to progressive hearing loss and impairs recovery from noise exposure in mice.

2021 ◽  
Author(s):  
Lisa S Nolan ◽  
Jing Chen ◽  
Ana Claudia Goncalves ◽  
Anwen Bullen ◽  
Karen P Steel ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Hair Cell ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stress Granules ◽  
Auditory Brainstem Responses ◽  
Progressive Hearing Loss ◽  
Granule Formation ◽  
Deficient Mice

Cell cycle associated protein 1 (Caprin1) is an RNA-binding protein that can regulate the cellular post-transcriptional response to stress. It is a component of both stress granules and neuronal RNA granules and is implicated in neurodegenerative disease, synaptic plasticity and long-term memory formation. Our previous work suggested that Caprin1 also plays a role in the response of the cochlea to stress. Here, targeted inner ear-deletion of Caprin1 in mice leads to an early onset, progressive hearing loss. Auditory brainstem responses from Caprin1-deficient mice show reduced thresholds, with a significant reduction in wave-I amplitudes compared to wildtype. Whilst hair cell structure and numbers were normal, the inner hair cell-spiral ganglion neuron (IHC-SGN) synapse revealed abnormally large post-synaptic GluA2 receptor puncta, a defect consistent with the observed wave-I reduction. Unlike wildtype mice, mild-noise induced hearing threshold shifts in Caprin1-deficient mice did not recover. Oxidative stress triggered TIA-1/HuR positive stress granule formation in ex-vivo cochlear explants from Caprin1-deficient mice, showing that stress granules could still be induced. Taken together, these findings suggest that Caprin1 plays a key role in maintenance of auditory function, where it regulates the normal status of the IHC-SGN synapse.

scholarly journals Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

PLoS ONE ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. e29683 ◽  
Author(s):  
Ryosuke Satoh ◽  
Akitomo Tanaka ◽  
Ayako Kita ◽  
Takahiro Morita ◽  
Yasuhiro Matsumura ◽  
...  
Keyword(s):  
Stress Response ◽  
Fission Yeast ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stress Granule ◽  
Granule Formation

scholarly journals Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication

2010 ◽  
Vol 84 (23) ◽  
pp. 12274-12284 ◽  
Author(s):  
Michael E. Lindquist ◽  
Aaron W. Lifland ◽  
Thomas J. Utley ◽  
Philip J. Santangelo ◽  
James E. Crowe
Keyword(s):  
Respiratory Syncytial Virus ◽  
Inclusion Bodies ◽  
Binding Protein ◽  
Mrna Translation ◽  
Stress Granules ◽  
Stress Granule ◽  
Granule Formation ◽  
Infected Cells ◽  
Viral Inclusion ◽  
Syncytial Virus

ABSTRACT Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.

scholarly journals RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome

2018 ◽  
Vol 115 (11) ◽  
pp. 2734-2739 ◽  
Author(s):  
Briana Van Treeck ◽  
David S. W. Protter ◽  
Tyler Matheny ◽  
Anthony Khong ◽  
Christopher D. Link ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Self Assembly ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stress Granules ◽  
Stress Granule ◽  
Local Concentration ◽  
Granule Formation ◽  
In Cells

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein–protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein–protein and RNA–RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA–RNA assembly in cells could lead to disease.

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