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Roy Parker - One of the best experts on this subject based on the ideXlab platform.
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rna self assembly contributes to Stress Granule formation and defining the Stress Granule transcriptome
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Briana Van Treeck, David S W Protter, Tyler Matheny, Anthony Khong, Christopher D Link, Roy ParkerAbstract: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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isolation of mammalian Stress Granule cores for rna seq analysis
Methods, 2017Co-Authors: Roy Parker, Tyler Matheny, Anthony Khong, Joshua R Wheeler, Saumya JainAbstract:Abstract Stress Granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular Stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian Stress Granules contain stable structures, referred to as “cores” that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from Stress Granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of Stress Granules and may assist in the discovery of the assembly mechanisms and functions of Stress Granules in a variety of biological contexts.
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isolation of yeast and mammalian Stress Granule cores
Methods, 2017Co-Authors: Joshua R Wheeler, Roy Parker, Anthony Khong, Saumya JainAbstract:Stress Granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian Stress Granules are comprised of two structures – an unstable shell and more stable cores. Herein we describe methodology for isolation of Stress Granule cores from both yeast and mammalian cells. The protocol consists of first enriching for Stress Granule cores using centrifugation and then further purifying Stress Granule cores using immunoprecipitation. The Stress Granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.
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distinct stages in Stress Granule assembly and disassembly
eLife, 2016Co-Authors: Joshua R Wheeler, Roy Parker, Tyler Matheny, Saumya Jain, Robert G AbrischAbstract: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.
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atpase modulated Stress Granules contain a diverse proteome and substructure
Cell, 2016Co-Authors: Saumya Jain, Roy Parker, Joshua R Wheeler, Robert W Walters, Anurag Agrawal, Anthony BarsicAbstract:Summary Stress Granules are mRNA-protein Granules that form when translation initiation is limited, and they are related to pathological Granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within Stress Granules that can be purified. Proteomic analysis of Stress Granule cores reveals a dense network of protein-protein interactions and links between Stress Granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved Stress Granule components. ATP is required for Stress Granule assembly and dynamics. Moreover, multiple ATP-driven machines affect Stress Granules differently, with the CCT complex inhibiting Stress Granule assembly, while the MCM and RVB complexes promote Stress Granule persistence. Our observations suggest that Stress Granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.
Paul Anderson - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into mammalian Stress Granule dynamics
Journal of Cell Biology, 2016Co-Authors: Marc D Panas, P A Ivanov, Paul AndersonAbstract:The accumulation of stalled translation preinitiation complexes (PICs) mediates the condensation of Stress Granules (SGs). Interactions between prion-related domains and intrinsically disordered protein regions found in SG-nucleating proteins promote the condensation of ribonucleoproteins into SGs. We propose that PIC components, especially 40S ribosomes and mRNA, recruit nucleators that trigger SG condensation. With resolution of Stress, translation reinitiation reverses this process and SGs disassemble. By cooperatively modulating the assembly and disassembly of SGs, ribonucleoprotein condensation can influence the survival and recovery of cells exposed to unfavorable environmental conditions.
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eif5a promotes translation elongation polysome disassembly and Stress Granule assembly
PLOS ONE, 2010Co-Authors: Takbum Ohn, P A Ivanov, Sarah Tisdale, Paul AndersonAbstract:Stress Granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental Stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and Stress Granule assembly. Time-course analysis reveals that this is due to a slowing of Stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of Stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and Stress Granule assembly in cells subjected to adverse environmental conditions.
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eukaryotic initiation factor 2α independent pathway of Stress Granule induction by the natural product pateamine a
Journal of Biological Chemistry, 2006Co-Authors: Yongjun Dang, Nancy Kedersha, Randal J. Kaufman, Daniel Romo, Myriam Gorospe, Paul AndersonAbstract:Abstract Stress Granules are aggregates of small ribosomal subunits, mRNA, and numerous associated RNA-binding proteins that include several translation initiation factors. Stress Granule assembly occurs in the cytoplasm of higher eukaryotic cells under a wide variety of Stress conditions, including heat shock, UV irradiation, hypoxia, and exposure to arsenite. Thus far, a unifying principle of eukaryotic initiation factor 2α phosphorylation prior to Stress Granule formation has been observed from the majority of experimental evidence. Pateamine A, a natural product isolated from marine sponge, was recently reported to inhibit eukaryotic translation initiation and induce the formation of Stress Granules. In this report, the protein composition and fundamental progression of Stress Granule formation and disassembly induced by pateamine A was found to be similar to that for arsenite. However, pateamine A-induced Stress Granules were more stable and less prone to disassembly than those formed in the presence of arsenite. Most significantly, pateamine A induced Stress Granules independent of eukaryotic initiation factor 2α phosphorylation, suggesting an alternative mechanism of formation from that previously described for other cellular Stresses. Taking into account the known inhibitory effect of pateamine A on eukaryotic translation initiation, a model is proposed to account for the induction of Stress Granules by pateamine A as well as other Stress conditions through perturbation of any steps prior to the rejoining of the 60S ribosomal subunit during the entire translation initiation process.
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Heme-regulated Inhibitor Kinase-mediated Phosphorylation of Eukaryotic Translation Initiation Factor 2 Inhibits Translation, Induces Stress Granule Formation, and Mediates Survival upon Arsenite Exposure
The Journal of biological chemistry, 2005Co-Authors: Edward Mcewen, Janejane Chen, Nancy Kedersha, Benbo Song, Donalyn Scheuner, Natalie Gilks, Anping Han, Paul Anderson, Randal J. KaufmanAbstract:Abstract Exposure to arsenite inhibits protein synthesis and activates multiple Stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of eukaryotic translation initiation factor 2 at Ser-51 on the α subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for Stress Granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2α, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for Stress Granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite Stress-induced phosphorylation of eukaryotic translation initiation factor 2α, inhibition of protein synthesis, Stress Granule formation, and survival.
Joshua R Wheeler - One of the best experts on this subject based on the ideXlab platform.
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isolation of mammalian Stress Granule cores for rna seq analysis
Methods, 2017Co-Authors: Roy Parker, Tyler Matheny, Anthony Khong, Joshua R Wheeler, Saumya JainAbstract:Abstract Stress Granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular Stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian Stress Granules contain stable structures, referred to as “cores” that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from Stress Granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of Stress Granules and may assist in the discovery of the assembly mechanisms and functions of Stress Granules in a variety of biological contexts.
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isolation of yeast and mammalian Stress Granule cores
Methods, 2017Co-Authors: Joshua R Wheeler, Roy Parker, Anthony Khong, Saumya JainAbstract:Stress Granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian Stress Granules are comprised of two structures – an unstable shell and more stable cores. Herein we describe methodology for isolation of Stress Granule cores from both yeast and mammalian cells. The protocol consists of first enriching for Stress Granule cores using centrifugation and then further purifying Stress Granule cores using immunoprecipitation. The Stress Granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.
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distinct stages in Stress Granule assembly and disassembly
eLife, 2016Co-Authors: Joshua R Wheeler, Roy Parker, Tyler Matheny, Saumya Jain, Robert G AbrischAbstract: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.
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atpase modulated Stress Granules contain a diverse proteome and substructure
Cell, 2016Co-Authors: Saumya Jain, Roy Parker, Joshua R Wheeler, Robert W Walters, Anurag Agrawal, Anthony BarsicAbstract:Summary Stress Granules are mRNA-protein Granules that form when translation initiation is limited, and they are related to pathological Granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within Stress Granules that can be purified. Proteomic analysis of Stress Granule cores reveals a dense network of protein-protein interactions and links between Stress Granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved Stress Granule components. ATP is required for Stress Granule assembly and dynamics. Moreover, multiple ATP-driven machines affect Stress Granules differently, with the CCT complex inhibiting Stress Granule assembly, while the MCM and RVB complexes promote Stress Granule persistence. Our observations suggest that Stress Granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.
Anthony Khong - One of the best experts on this subject based on the ideXlab platform.
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rna self assembly contributes to Stress Granule formation and defining the Stress Granule transcriptome
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Briana Van Treeck, David S W Protter, Tyler Matheny, Anthony Khong, Christopher D Link, Roy ParkerAbstract: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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isolation of mammalian Stress Granule cores for rna seq analysis
Methods, 2017Co-Authors: Roy Parker, Tyler Matheny, Anthony Khong, Joshua R Wheeler, Saumya JainAbstract:Abstract Stress Granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular Stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian Stress Granules contain stable structures, referred to as “cores” that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from Stress Granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of Stress Granules and may assist in the discovery of the assembly mechanisms and functions of Stress Granules in a variety of biological contexts.
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isolation of yeast and mammalian Stress Granule cores
Methods, 2017Co-Authors: Joshua R Wheeler, Roy Parker, Anthony Khong, Saumya JainAbstract:Stress Granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian Stress Granules are comprised of two structures – an unstable shell and more stable cores. Herein we describe methodology for isolation of Stress Granule cores from both yeast and mammalian cells. The protocol consists of first enriching for Stress Granule cores using centrifugation and then further purifying Stress Granule cores using immunoprecipitation. The Stress Granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.
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disruption of Stress Granule formation by the multifunctional cricket paralysis virus 1a protein
Journal of Virology, 2017Co-Authors: Anthony Khong, Craig H Kerr, Arabinda Nayak, Clarence H L Yeung, Kathleen Keatings, Douglas W Allan, Eric JanAbstract:Stress Granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular Stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called Stress Granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.
Saumya Jain - One of the best experts on this subject based on the ideXlab platform.
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isolation of mammalian Stress Granule cores for rna seq analysis
Methods, 2017Co-Authors: Roy Parker, Tyler Matheny, Anthony Khong, Joshua R Wheeler, Saumya JainAbstract:Abstract Stress Granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular Stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian Stress Granules contain stable structures, referred to as “cores” that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from Stress Granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of Stress Granules and may assist in the discovery of the assembly mechanisms and functions of Stress Granules in a variety of biological contexts.
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isolation of yeast and mammalian Stress Granule cores
Methods, 2017Co-Authors: Joshua R Wheeler, Roy Parker, Anthony Khong, Saumya JainAbstract:Stress Granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian Stress Granules are comprised of two structures – an unstable shell and more stable cores. Herein we describe methodology for isolation of Stress Granule cores from both yeast and mammalian cells. The protocol consists of first enriching for Stress Granule cores using centrifugation and then further purifying Stress Granule cores using immunoprecipitation. The Stress Granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.
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distinct stages in Stress Granule assembly and disassembly
eLife, 2016Co-Authors: Joshua R Wheeler, Roy Parker, Tyler Matheny, Saumya Jain, Robert G AbrischAbstract: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.
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atpase modulated Stress Granules contain a diverse proteome and substructure
Cell, 2016Co-Authors: Saumya Jain, Roy Parker, Joshua R Wheeler, Robert W Walters, Anurag Agrawal, Anthony BarsicAbstract:Summary Stress Granules are mRNA-protein Granules that form when translation initiation is limited, and they are related to pathological Granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within Stress Granules that can be purified. Proteomic analysis of Stress Granule cores reveals a dense network of protein-protein interactions and links between Stress Granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved Stress Granule components. ATP is required for Stress Granule assembly and dynamics. Moreover, multiple ATP-driven machines affect Stress Granules differently, with the CCT complex inhibiting Stress Granule assembly, while the MCM and RVB complexes promote Stress Granule persistence. Our observations suggest that Stress Granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.
