ABSTRACT
The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.
Subject(s)
Cytoplasmic Granules/metabolism , DNA Helicases/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Ribonucleoproteins/metabolism , Cell Line, Tumor , Cytoplasm/metabolism , Cytoplasmic Structures/metabolism , HEK293 Cells , Humans , Phosphorylation , RNA/metabolismABSTRACT
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Dipeptides/metabolism , Frontotemporal Dementia/metabolism , Proteins/metabolism , RNA-Binding Proteins/metabolism , Amyotrophic Lateral Sclerosis/genetics , Animals , C9orf72 Protein , Cell Nucleolus/metabolism , Cytoplasmic Granules/metabolism , DNA Repeat Expansion , Dipeptides/genetics , Drosophila melanogaster/genetics , Frontotemporal Dementia/genetics , Humans , Intracellular Membranes/metabolism , Nuclear Pore/metabolism , Peptides/genetics , Peptides/metabolism , Proteins/geneticsABSTRACT
Stress granules and P bodies are conserved cytoplasmic aggregates of nontranslating messenger ribonucleoprotein complexes (mRNPs) implicated in the regulation of mRNA translation and decay and are related to RNP granules in embryos, neurons, and pathological inclusions in some degenerative diseases. Using baker's yeast, 125 genes were identified in a genetic screen that affected the dynamics of P bodies and/or stress granules. Analyses of such mutants, including CDC48 alleles, provide evidence that stress granules can be targeted to the vacuole by autophagy, in a process termed granulophagy. Moreover, stress granule clearance in mammalian cells is reduced by inhibition of autophagy or by depletion or pathogenic mutations in valosin-containing protein (VCP), the human ortholog of CDC48. Because mutations in VCP predispose humans to amyotrophic lateral sclerosis, frontotemporal lobar degeneration, inclusion body myopathy, and multisystem proteinopathy, this work suggests that autophagic clearance of stress granule related and pathogenic RNP granules that arise in degenerative diseases may be important in reducing their pathology.
Subject(s)
Adenosine Triphosphatases/metabolism , Autophagy , Cell Cycle Proteins/metabolism , Cytoplasmic Granules/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/genetics , Animals , Cell Cycle Proteins/genetics , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Humans , Mice , Mutation , RNA Stability , RNA, Messenger/metabolism , Ribonucleoproteins/genetics , Saccharomyces cerevisiae Proteins , Valosin Containing ProteinABSTRACT
Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.
Subject(s)
Cell Cycle Proteins/metabolism , Cytoplasmic Granules/metabolism , Intrinsically Disordered Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Stress, Physiological , Ubiquitins/metabolism , Adaptor Proteins, Signal Transducing , Autophagy-Related Proteins , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Female , HeLa Cells , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Models, Molecular , Protein Aggregation, Pathological , Protein Binding , Protein Conformation , Protein Domains , Protein Folding , Structure-Activity Relationship , Ubiquitins/chemistry , Ubiquitins/geneticsABSTRACT
Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Arginine/metabolism , Cytoplasmic Granules/metabolism , Dipeptides/metabolism , Intrinsically Disordered Proteins/metabolism , Proteins/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Arginine/chemistry , C9orf72 Protein , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cytoplasmic Granules/pathology , DNA Helicases , Dipeptides/chemistry , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , HeLa Cells , Humans , Intrinsically Disordered Proteins/chemistry , Lipid Droplets/metabolism , Phosphorylation , Poly-ADP-Ribose Binding Proteins , Protein Domains , Proteins/chemistry , RNA/metabolism , RNA Helicases , RNA Recognition Motif Proteins , Time Factors , TransfectionABSTRACT
Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies.
Subject(s)
Cell Nucleus/metabolism , Macromolecular Substances/metabolism , Nuclear Proteins/metabolism , Protein Multimerization , Repressor Proteins/metabolism , Humans , Kruppel-Like Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Domains , Ubiquitination , Zinc Finger Protein Gli3ABSTRACT
Adult-onset inherited myopathies with similar pathological features, including hereditary inclusion body myopathy (hIBM) and limb-girdle muscular dystrophy (LGMD), are a genetically heterogeneous group of muscle diseases. It is unclear whether these inherited myopathies initiated by mutations in distinct classes of genes are etiologically related. Here, we exploit a genetic model system to establish a mechanistic link between diseases caused by mutations in two distinct genes, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila melanogaster homologs of human hnRNPA2B1 and DNAJB6, respectively. We introduced disease-homologous mutations to Hrb98DE, thus capturing mutation-dependent phenotypes in a genetically tractable model system. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins (RBPs), suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover, wild-type, but not disease-associated, mutant forms of MRJ interacted with RBPs after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interactions between disease-linked RBPs and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.
Subject(s)
Contracture/congenital , Drosophila melanogaster/genetics , HSP40 Heat-Shock Proteins/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Molecular Chaperones/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Myositis, Inclusion Body/congenital , Nerve Tissue Proteins/genetics , Ophthalmoplegia/genetics , Adult , Age of Onset , Amino Acid Sequence , Animals , Contracture/genetics , Contracture/metabolism , Contracture/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Regulation , HSP40 Heat-Shock Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Molecular Chaperones/metabolism , Molecular Sequence Data , Muscles/metabolism , Muscles/pathology , Muscular Dystrophies, Limb-Girdle/metabolism , Muscular Dystrophies, Limb-Girdle/pathology , Mutation , Myositis, Inclusion Body/genetics , Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Nerve Tissue Proteins/metabolism , Ophthalmoplegia/metabolism , Ophthalmoplegia/pathology , Phenotype , Protein Binding , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial amyotrophic lateral sclerosis (ALS). Profilin 1 is a well studied actin-binding protein but how PFN1 mutations cause ALS is unknown. The budding yeast, Saccharomyces cerevisiae, has one PFN1 ortholog. We expressed the ALS-linked profilin 1 mutant proteins in yeast, demonstrating a loss of protein stability and failure to restore growth to profilin mutant cells, without exhibiting gain-of-function toxicity. This model provides for simple and rapid screening of novel ALS-linked PFN1 variants. To gain insight into potential novel roles for profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with yeast cells lacking profilin (pfy1Δ). Unexpectedly, deletion of several stress granule and processing body genes, including pbp1Δ, were found to be synthetic lethal with pfy1Δ. Mutations in ATXN2, the human ortholog of PBP1, are a known ALS genetic risk factor and ataxin 2 is a stress granule component in mammalian cells. Given this genetic interaction and recent evidence linking stress granule dynamics to ALS pathogenesis, we hypothesized that profilin 1 might also associate with stress granules. Here we report that profilin 1 and related protein profilin 2 are novel stress granule-associated proteins in mouse primary cortical neurons and in human cell lines and that ALS-linked mutations in profilin 1 alter stress granule dynamics, providing further evidence for the potential role of stress granules in ALS pathogenesis.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Cytoplasmic Granules/metabolism , Mutation/genetics , Oxidative Stress/genetics , Profilins/genetics , Animals , Arsenites/pharmacology , Ataxins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , Cycloheximide/pharmacology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/genetics , DNA Helicases , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Eukaryotic Initiation Factor-1/metabolism , Humans , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Oxidative Stress/drug effects , Poly-ADP-Ribose Binding Proteins , Protein Synthesis Inhibitors/pharmacology , RNA Helicases , RNA Recognition Motif Proteins , Teratogens/pharmacology , Two-Hybrid System TechniquesABSTRACT
The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (ß) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2ß subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2ß subunit, that also possess the conserved features of regulatory CK2ß subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2ß proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.
Subject(s)
Casein Kinase II/genetics , Casein Kinase II/isolation & purification , Ceratitis capitata/enzymology , Ceratitis capitata/genetics , Protein Subunits/genetics , Protein Subunits/isolation & purification , Recombinant Proteins/isolation & purification , Amino Acid Sequence , Animals , Casein Kinase II/chemistry , Catalytic Domain , Chromatography, Affinity , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Immobilized Proteins/metabolism , Molecular Sequence Data , Protein Subunits/chemistry , Recombinant Proteins/genetics , Sequence AlignmentABSTRACT
Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulumassociated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granulespecific interaction network, resulting in granule disassembly.
Subject(s)
Blood Proteins/metabolism , Cytoplasmic Granules/metabolism , DNA Helicases/metabolism , Heat-Shock Response , Membrane Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/metabolism , Autophagy , Cell Line, Tumor , DNA Helicases/chemistry , DNA Helicases/genetics , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Intracellular Membranes/metabolism , Mutation , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/genetics , Polyubiquitin/metabolism , Protein Domains , Proteolysis , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Recognition Motif Proteins/chemistry , RNA Recognition Motif Proteins/genetics , Ubiquitinated Proteins/chemistry , UbiquitinationABSTRACT
Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget's disease, or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.