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.

scholarly journals Pak1 kinase regulates ribonucleoprotein granules through Sts5 for polarized growth and the glucose starvation response

2021 ◽  
Author(s):  
Joseph O. Magliozzi ◽  
James B. Moseley
Keyword(s):  
Cell Shape ◽  
Rna Binding ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Glucose Starvation ◽  
Polarized Growth ◽  
P Bodies ◽  
Intrinsically Disordered ◽  
Rapid Dissolution ◽  
Rnp Granules

ABSTRACTFission 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 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 increase P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to P bodies with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to these ribonucleoprotein (RNP) granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through RNPs during normal and stressed growth conditions.

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 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 RNA Binding Protein TAF15 Suppresses Toxicity in a Yeast Model of FUS Proteinopathy

2021 ◽  
Author(s):  
Elliott Hayden ◽  
Aicha Kebe ◽  
Shuzhen Chen ◽  
Abagail Chumley ◽  
Chenyi Xia ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stress Granules ◽  
Rna Recognition Motif ◽  
Body Formation ◽  
P Bodies ◽  
Associated Proteins ◽  
Rnp Granules ◽  
Yeast Model

Abstract Mutations in Fused in Sarcoma (FUS), an RNA binding protein that functions in multiple steps in gene expression regulation and RNA processing, are known to cause familial amyotrophic lateral sclerosis (ALS). Since this discovery, mutations in several other RNA binding proteins (RBPs) have also been linked to ALS. Some of these ALS-associated RBPs have been shown to colocalize with ribonucleoprotein (RNP) granules such as stress granules and processing bodies (p-bodies). Characterization of ALS-associated proteins, their mis-localization, aggregation and toxicity in cellular and animal models have provided critical insights in disease. More and more evidence has emerged supporting a hypothesis that impaired clearance, inappropriate assembly, and dysregulation of RNP granules play a role in ALS. Through genome-scale overexpression screening of a yeast model of FUS toxicity, we found that TAF15, a human RBP with a similar protein domain structure and belonging to the same FET protein family as FUS, suppresses FUS toxicity. The suppressor effect of TAF15 is specific to FUS and not found in other yeast models of neurodegenerative disease-associated proteins. We showed that the RNA recognition motif (RRM) of TAF15 is required for its rescue of FUS toxicity. Furthermore, FUS and TAF15 physically interact, and the C-terminus of TAF15 is required for both the physical protein-protein interaction and its protection against FUS toxicity. Finally, while FUS induces and colocalizes with both stress granules and p-bodies, TAF15 only induces and colocalizes with p-bodies. Importantly, co-expression of FUS and TAF15 induces more p-bodies than individually expressing each gene alone, and FUS toxicity is exacerbated in yeast that is deficient in p-body formation. Overall, our findings suggest a role of p-body formation in the suppression of FUS toxicity by TAF15.

scholarly journals Age-induced P-bodies become detrimental and shorten the lifespan of yeast

2021 ◽  
Author(s):  
Joonhyuk Choi ◽  
Shuhao Wang ◽  
Yang Li ◽  
Nan Hao ◽  
Brian M Zid
Keyword(s):  
Irreversible Process ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Model System ◽  
Yeast Cells ◽  
P Bodies ◽  
Daughter Cells ◽  
Progressive Loss ◽  
External Stresses ◽  
Mother Cells

Aging is an irreversible process characterized by a progressive loss of homeostasis in cells, which often manifests as protein aggregates. Recently, it has been speculated that aggregates of RNA-binding proteins (RBPs) may go through pathological transitions during aging and drive the progression of age-associated neurodegenerative diseases. Using Saccharomyces cerevisiae as a model system of aging, we find that P-bodies - an RBP granule that is formed and can be beneficial for cell growth during stress conditions - naturally form during aging without any external stresses and an increase in P-body intensity is negatively correlated with the future lifespan of yeast cells. When mother cells transfer age-induced P-bodies to daughter cells, the mother cells extend lifespan, while the daughter cells grow poorly, suggesting that these age-induced P-bodies may be directly pathological. Furthermore, we find that suppressing acidification of the cytosol during aging slows down the increase in the intensity of P-body foci and extends lifespan. Our data suggest that acidification of the cytosol may facilitate the pathological transition of RBP granules during aging.

scholarly journals eIF3a Destabilization and TDP-43 Alter Dynamics of Heat-Induced Stress Granules

2021 ◽  
Vol 22 (10) ◽  
pp. 5164
Author(s):  
Ivana Malcova ◽  
Lenka Senohrabkova ◽  
Lenka Novakova ◽  
Jiri Hasek
Keyword(s):  
Stress Granules ◽  
Stress Relief ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Translation Arrest ◽  
P Bodies ◽  
Bona Fide ◽  
Lower Temperature ◽  
Lateral Sclerosis

Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.

scholarly journals Hsp90 Regulates the Function of Argonaute 2 and Its Recruitment to Stress Granules and P-Bodies

2009 ◽  
Vol 20 (14) ◽  
pp. 3273-3284 ◽  
Author(s):  
Justin M. Pare ◽  
Nasser Tahbaz ◽  
Joaquín López-Orozco ◽  
Paul LaPointe ◽  
Paul Lasko ◽  
...  
Keyword(s):  
Rna Binding ◽  
Stress Granules ◽  
Processing Bodies ◽  
P Bodies ◽  
Argonaute 2 ◽  
Ribonucleoprotein Complexes ◽  
Protein Activator ◽  
Hsp 90 ◽  
Interfering Rna ◽  
Expression Processing

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNA-activated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs.

scholarly journals Asc1p, a WD40-domain containing adaptor protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes

2004 ◽  
Vol 380 (3) ◽  
pp. 823-830 ◽  
Author(s):  
Sonja BAUM ◽  
Margarethe BITTINS ◽  
Steffen FREY ◽  
Matthias SEEDORF
Keyword(s):  
Rna Binding ◽  
Elongation Factor ◽  
Adaptor Protein ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Free Form ◽  
Dependent Manner ◽  
Signalling Molecules ◽  
C Terminus ◽  
Specific Mrnas

Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein–protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys1067 at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs.

scholarly journals Antiviral Protein APOBEC3G Localizes to Ribonucleoprotein Complexes Found in P Bodies and Stress Granules

2006 ◽  
Vol 81 (5) ◽  
pp. 2165-2178 ◽  
Author(s):  
Sarah Gallois-Montbrun ◽  
Beatrice Kramer ◽  
Chad M. Swanson ◽  
Helen Byers ◽  
Steven Lynham ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Affinity Purification ◽  
Stress Granules ◽  
Antiviral Protein ◽  
Cellular Targets ◽  
P Bodies ◽  
Ribonucleoprotein Complexes ◽  
Protein Components

ABSTRACT Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function.

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