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

Rapid Dissolution ◽

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.

Pak1 kinase controls cell shape through ribonucleoprotein granules

eLife ◽

10.7554/elife.67648 ◽

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 ◽

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.

The CDC42 Homolog of the Dimorphic FungusPenicillium marneffei Is Required for Correct Cell Polarization during Growth but Not Development

Journal of Bacteriology ◽

10.1128/jb.183.11.3447-3457.2001 ◽

2001 ◽

Vol 183 (11) ◽

pp. 3447-3457 ◽

Cited By ~ 63

Author(s):

Kylie J. Boyce ◽

Michael J. Hynes ◽

Alex Andrianopoulos

Keyword(s):

Cell Shape ◽

Pathogenic Fungus ◽

Cell Polarization ◽

Dominant Negative ◽

Yeast Cells ◽

Polarized Growth ◽

Temperature Dependent ◽

Human Pathogenic Fungus ◽

Rho Family

ABSTRACT The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffeinamed cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflAfunction is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression ofcflA alleles in Aspergillus nidulansprevented conidiation.

Proteomic analysis of Dhh1 complexes reveals a role for Hsp40 chaperone Ydj1 in yeast P-body assembly

10.1101/016402 ◽

2015 ◽

Author(s):

Gregory A. Cary ◽

Dani B.N. Vinh ◽

Patrick May ◽

Rolf Kuestner ◽

Aimee M. Dudley

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Active Role ◽

Low Complexity ◽

Mitochondrial Rna ◽

P Bodies ◽

Glucose Depletion ◽

Catalytic Rnas ◽

P-bodies (PB) are ribonucleoprotein (RNP) complexes that aggregate into cytoplasmic foci when cells are exposed to stress. While the conserved mRNA decay and translational repression machineries are known components of PB, how and why cells assemble RNP complexes into large foci remain unclear. Using mass spectrometry to analyze proteins immunoisolated with the core PB protein Dhh1, we show that a considerable number of proteins contain low-complexity (LC) sequences, similar to proteins highly represented in mammalian RNP granules. We also show that the Hsp40 chaperone Ydj1, which contains an LC domain and controls prion protein aggregation, is required for the formation of Dhh1-GFP foci upon glucose depletion. New classes of proteins that reproducibly co-enrich with Dhh1-GFP during PB induction include proteins involved in nucleotide or amino acid metabolism, glycolysis, tRNA aminoacylation, and protein folding. Many of these proteins have been shown to form foci in response to other stresses. Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins, suggesting an active role for RNA in PB function. Thus, global characterization of PB composition has uncovered proteins and RNA that are important for PB assembly.

ANTAGONISTIC ROLES FOR ATAXIN-2 STRUCTURED AND DISORDERED DOMAINS IN RNP CONDENSATION

10.1101/2020.07.02.184796 ◽

2020 ◽

Author(s):

Amanjot Singh ◽

Joern Huelsmeier ◽

Arvind Reddy Kandi ◽

Sai Shruti Pothapragada ◽

Jens Hillebrand ◽

...

Keyword(s):

Translational Control ◽

Rna Binding ◽

Rna Binding Proteins ◽

Biological Significance ◽

Spinocerebellar Ataxia Type ◽

Granule Formation ◽

Target Mrnas ◽

Ataxin 2 ◽

ABSTRACTAtaxin-2 is a conserved translational control protein associated with spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS) as well as an important target for ALS therapeutics under development. Despite its clinical and biological significance, Ataxin-2’s activities, mechanisms and functions are not well understood. While Drosophila Ataxin-2 (Atx2) mediates mRNP condensation via a C-terminal intrinsically disordered domain (cIDR), how Ataxin-2 IDRs work with structured (Lsm, Lsm-AD and PAM2) domains to enable positive and negative regulation of target mRNAs remains unclear. Using TRIBE (Targets of RNA-Binding Proteins Identified by Editing) technology, we identified and analysed Atx-2 target mRNAs in the Drosophila brain. We show that Atx2 preferentially interacts with AU-rich elements (AREs) in 3’UTRs and plays a broad role in stabilization of identified target mRNAs. Strikingly, Atx2 interaction with its targets is dependent on the cIDR domain required for neuronal-granule formation. In contrast, Atx2 lacking its Lsm domain not only interacts more efficiently with the target mRNA identified, but also forms larger RNP granules. Providing an extensive dataset of Atx2-interacting brain mRNAs, our results demonstrate that Atx2: (a) interacts with target mRNAs within RNP granules; (b) modulates the turnover of these target mRNAs; (c) has an additional essential role outside of mRNP granules; and (d) contains distinct protein domains that drive or oppose RNP-granule assembly. These findings increase understanding of neuronal translational control mechanisms and inform Ataxin-2-based interventions in development for SCA2 and ALS.

RNA Droplets

Annual Review of Biophysics ◽

10.1146/annurev-biophys-052118-115508 ◽

2020 ◽

Vol 49 (1) ◽

pp. 247-265 ◽

Cited By ~ 10

Author(s):

Kevin Rhine ◽

Velinda Vidaurre ◽

Sua Myong

Keyword(s):

Phase Separation ◽

Intrinsically Disordered Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Disordered Proteins ◽

In Vivo Studies ◽

Anionic Polymer ◽

Temporal And Spatial Distribution ◽

Liquid–liquid phase separation is emerging as the universal mechanism by which membraneless cellular granules form. Despite many previous studies on condensation of intrinsically disordered proteins and low complexity domains, we lack understanding about the role of RNA, which is the essential component of all ribonucleoprotein (RNP) granules. RNA, as an anionic polymer, is inherently an excellent platform for achieving multivalency and can accommodate many RNA binding proteins. Recent findings have highlighted the diverse function of RNA in tuning phase-separation propensity up or down, altering viscoelastic properties and thereby driving immiscibility between different condensates. In addition to contributing to the biophysical properties of droplets, RNA is a functionally critical constituent that defines the identity of cellular condensates and controls the temporal and spatial distribution of specific RNP granules. In this review, we summarize what we have learned so far about such roles of RNA in the context of in vitro and in vivo studies.

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Molecular Biology of the Cell ◽

10.1091/mbc.3.9.1025 ◽

1992 ◽

Vol 3 (9) ◽

pp. 1025-1035 ◽

Cited By ~ 41

Author(s):

K Madden ◽

M Snyder

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Types ◽

Growth Conditions ◽

Yeast Cells ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Polarized Growth ◽

Mating Partner ◽

Polarized Cell Growth ◽

External Conditions

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.

Numerous interactions act redundantly to assemble a tunable size of P bodies in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712396114 ◽

2017 ◽

Vol 114 (45) ◽

pp. E9569-E9578 ◽

Cited By ~ 44

Author(s):

Bhalchandra S. Rao ◽

Roy Parker

Keyword(s):

Protein Interactions ◽

Growth Conditions ◽

Protein Protein Interactions ◽

Protein Assemblies ◽

P Bodies ◽

Multiple Protein ◽

Body Components ◽

Rnp Granules ◽

General Translation ◽

Liquid Liquid Phase Separation

Eukaryotic cells contain multiple RNA–protein assemblies referred to as RNP granules, which are thought to form through multiple protein–protein interactions analogous to a liquid–liquid phase separation. One class of RNP granules consists of P bodies, which consist of nontranslating mRNAs and the general translation repression and mRNA degradation machinery. P bodies have been suggested to form predominantly through interactions of Edc3 and a prion-like domain on Lsm4. In this work, we provide evidence that P-body assembly can be driven by multiple different protein–protein and/or protein–RNA interactions, including interactions involving Dhh1, Psp2, and Pby1. Moreover, the relative importance of specific interactions can vary with different growth conditions. Based on these observations, we develop a summative model wherein the P-body assembly phenotype of a given mutant can be predicted from the number of currently known protein–protein interactions between P-body components.

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

10.21203/rs.3.rs-437201/v1 ◽

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.

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

10.1101/2021.11.05.467477 ◽

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.

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

Biochemical Journal ◽

10.1042/bj20031962 ◽

2004 ◽

Vol 380 (3) ◽

pp. 823-830 ◽

Cited By ~ 73

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.