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 m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins

2019 ◽  
Author(s):  
Ye Fu ◽  
Xiaowei Zhuang
Keyword(s):  
Phase Separation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Stress Granule ◽  
Granule Formation ◽  
Intrinsically Disordered ◽  
Condensate Formation ◽  
Resolution Imaging

AbstractDiverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.

scholarly journals P bodies promote stress granule assembly in Saccharomyces cerevisiae

2008 ◽  
Vol 183 (3) ◽  
pp. 441-455 ◽  
Author(s):  
J. Ross Buchan ◽  
Denise Muhlrad ◽  
Roy Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Protein Composition ◽  
Stress Granules ◽  
Glucose Deprivation ◽  
Stress Granule ◽  
Granule Formation ◽  
P Bodies ◽  
Rna Granules ◽  
Kinetics Of

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.

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 Distinct stages in stress granule assembly and disassembly

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Joshua R Wheeler ◽  
Tyler Matheny ◽  
Saumya Jain ◽  
Robert Abrisch ◽  
Roy Parker
Keyword(s):  
Time Course ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stress Granules ◽  
Stress Granule ◽  
Early Event ◽  
Intrinsically Disordered

Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.

scholarly journals OptoGranules reveal the evolution of stress granules to ALS-FTD pathology

2018 ◽  
Author(s):  
Peipei Zhang ◽  
Baochang Fan ◽  
Peiguo Yang ◽  
Jamshid Temirov ◽  
James Messing ◽  
...  
Keyword(s):  
Material Properties ◽  
Rna Binding ◽  
Chimeric Protein ◽  
Stress Granules ◽  
Stress Granule ◽  
Cytoplasmic Inclusions ◽  
Intrinsically Disordered ◽  
Biological Studies ◽  
Cryptochrome 2 ◽  
As Stress

Stress granules are non-membranous assemblies of mRNA and protein that form in response to a variety of stressors. Genetic, pathologic, biophysical and cell biological studies have implicated disturbances in the dynamics of membrane-less organelles, such as stress granules, as a pathobiological component of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1–12. This confluence of evidence has inspired the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of stress granules; however, this concept has remained largely untestable in available models of stress granule assembly, which require the confounding variable of exogenous stressors. Here we demonstrate the development and use of a light-inducible stress granule system, termed OptoGranules, which permits discrete, experimental control of the dynamics and material properties of stress granules in living cells in the absence of exogenous stressors. The nucleator in this system is Opto-G3BP1, a light-sensitive chimeric protein assembled from the intrinsically disordered region (IDR) and RNA-binding domain of G3BP1 combined with the light-sensitive oligomerization domain of Arabidopsis thaliana cryptochrome 2 (CRY2) photolyase homology region (PHR). Upon stimulation with blue light, Opto-G3BP1 initiates the rapid assembly of dynamic, cytoplasmic, liquid granules that are composed of canonical stress granule components, including G3BP1, PABP, TIA1, TIAR, eIF4G, eIF3η, ataxin 2, GLE1, TDP-43 and polyadenylated RNA. With this system, we demonstrate that persistent or repetitive assembly of stress granules is cytotoxic and is accompanied by the evolution of stress granules to neuronal cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.

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 Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae

2010 ◽  
Vol 189 (5) ◽  
pp. 813-827 ◽  
Author(s):  
Je-Hyun Yoon ◽  
Eui-Ju Choi ◽  
Roy Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Interactions ◽  
Messenger Rna ◽  
Mrna Decay ◽  
Control Point ◽  
Stress Granules ◽  
Mrna Degradation ◽  
Stress Granule ◽  
Granule Formation ◽  
P Bodies

Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping.

scholarly journals DAZL is essential for stress granule formation implicated in germ cell survival upon heat stress

Development ◽  
2012 ◽  
Vol 139 (3) ◽  
pp. 568-578 ◽  
Author(s):  
B. Kim ◽  
H. J. Cooke ◽  
K. Rhee
Keyword(s):  
Heat Stress ◽  
Germ Cell ◽  
Cell Survival ◽  
Stress Granule ◽  
Granule Formation

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 Pak1 kinase controls cell shape through ribonucleoprotein granules

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Joseph O Magliozzi ◽  
James B Moseley
Keyword(s):  
Cell Shape ◽  
Rna Binding ◽  
Stress Granules ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Glucose Starvation ◽  
Glucose Addition ◽  
P Bodies ◽  
Intrinsically Disordered ◽  
Rapid Dissolution

Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.

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