Osmotic stress-induced, rapid clustering of IGF2BP proteins nucleates stress granule assembly (original) (raw)
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Stress granules are dispensable for mRNA stabilization during cellular stress
Nucleic acids research, 2015
During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting ...
Macromolecular Crowding Regulates Assembly of mRNA Stress Granules after Osmotic Stress
Journal of Biological Chemistry
The massive uptake of compatible osmolytes such as betaine, taurine, and myo-inositol is a protective response shared by all eukaryotes exposed to hypertonic stress. Their accumulation results mostly from the expression of specific transporters triggered by the transcriptional factor NFAT5/TonEBP. This allows the recovery of the cell volume without increasing intracellular ionic strength. In this study we consider the assembly and dissociation of mRNA stress granules (SGs) in hypertonic-stressed cells and the role of compatible osmolytes. In agreement with in vitro results obtained on isolated mRNAs, both macromolecular crowding and a high ionic strength favor the assembly of SGs in normal rat kidney epithelial cells. However, after hours of constant hypertonicity, the slow accumulation in the cytoplasm of compatible osmolytes via specific transporters both reduces macromolecular crowding and ionic strength, thus leading to the progressive dissociation of SGs. In line with this, whe...
Single-molecule imaging reveals translation of mRNAs localized to stress granules
2020
SUMMARYCellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation.
Dynamic Shuttling of Tia-1 Accompanies the Recruitment of mRNA to Mammalian Stress Granules
Journal of Cell Biology, 2000
Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein-tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1 ⌬ RRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.
Journal of Biological Chemistry, 2012
The intracellular accumulation of compatible osmolytes in hypertonic conditions reduces macromolecular crowding and ionic strength. Results: Compatible osmolytes disassemble mRNA stress granules. Hypertonic-preconditioning and gap-junction communication favor cell survival via compatible osmolyte accumulation. Conclusion: Macromolecular crowding regulates stress granule assembly and, thus, the cell fate after osmotic stress. Significance: Compatible osmolytes can promote cell survival through their action on stress granules.
Dynamics of mRNA entry into stress granules
Nature Cell Biology, 2019
Stressed eukaryotic cells store mRNAs in protein-rich condensates called stress granules. Using single-molecule tracking techniques to examine how mRNAs enter stress granules, a new study shows that mRNAs make transient contacts with the granule surface before stable association, and become largely immobile after entry.
Journal of Biological Chemistry, 2009
Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only ϳ10 -20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.
Principles of Stress Granules Revealed by Imaging Approaches
Cold Spring Harbor Perspectives in Biology
Eukaryotic cells contain a large number of RNA-protein assemblies, generically referred to as ribonucleoprotein (RNP) granules. Such RNP granules include stress granules and P-bodies in the cytosol and the nucleolus, Cajal bodies, and paraspeckles in the nucleus. A variety of imaging approaches have been used to reveal different components, structural features, and dynamics of RNP granules. In this review, we discuss imaging approaches that have been used to study stress granules and the insights gained from these experiments. A general theme is that these approaches can be transferred to other RNP granules to examine similar aspects of their composition, ultrastructure, dynamics and control. Outline 1 Introduction 2 Protein components of stress granules 3 RNA components of stress granules 4 Microscopic analyses of stress granules reveal internal ultrastructure 5 Imaging of stress granule assembly and disassembly in real time 6 Stress granules are dynamic assemblies 7 Perspectives and future directions References
PLOS Genetics, 2012
Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stressinduced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.
Role of Microtubules in Stress Granule Assembly
Journal of Biological Chemistry, 2009
Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only ϳ10-20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.