Stress Granule Disassembly Research Articles

Decoding the interplay between m6A modification and stress granule stability by live-cell imaging.

N6-methyladenosine (m6A)-modified mRNAs and their cytoplasmic reader YTHDFs are colocalized with stress granules (SGs) under stress conditions, but the interplay between m6A modification and SG stability remains unclear. Here, we presented a spatiotemporal m6A imaging system (SMIS) that can monitor the m6A modification and the translation of mRNAs with high specificity and sensitivity in a single live cell. SMIS showed that m6A-modified reporter mRNAs dynamically enriched into SGs under arsenite stress and gradually partitioned into the cytosol as SG disassembled. SMIS revealed that knockdown of YTHDF2 contributed to SG disassembly, resulting in the fast redistribution of mRNAs from SGs and rapid recovery of stalled translation. The mechanism is that YTHDF2 can regulate SG stability through the interaction with G3BP1 in m6A-modified RNA-dependent manner. Our results suggest a mechanism for the interplay between m6A modification and SG through YTHDF2 regulation.

Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties.

The human genome is replete with sequences derived from foreign elements including endogenous retrovirus-like proteins of unknown function. Here we show that UBQLN2, a ubiquitin-proteasome shuttle factor implicated in neurodegenerative diseases, is regulated by the linked actions of two retrovirus-like proteins, RTL8 and PEG10. RTL8 confers on UBQLN2 the ability to complex with and regulate PEG10. PEG10, a core component of stress granules, drives the recruitment of UBQLN2 to stress granules under various stress conditions, but can only do so when RTL8 is present. Changes in PEG10 levels further remodel the kinetics of stress granule disassembly and overall composition by incorporating select extracellular vesicle proteins. Within stress granules, PEG10 forms virus-like particles, underscoring the structural heterogeneity of this class of biomolecular condensates. Together, these results reveal an unexpected link between pathways of cellular proteostasis and endogenous retrovirus-like proteins.

µMap proximity labeling in living cells reveals stress granule disassembly mechanisms.

Phase-separated condensates are membrane-less intracellular structures comprising dynamic protein interactions that organize essential biological processes. Understanding the composition and dynamics of these organelles advances our knowledge of cellular behaviors and disease pathologies related to granule dysregulation. In this study, we apply microenvironment mapping with a HaloTag-based platform (HaloMap) to characterize intracellular stress granule dynamics in living cells. After validating the robustness and sensitivity of this approach, we then profile the stress granule proteome throughout the formation and disassembly and under pharmacological perturbation. These experiments reveal several ubiquitin-related modulators, including the HECT (homologous to E6AP C terminus) E3 ligases ITCH and NEDD4L, as well as the ubiquitin receptor toll-interacting protein TOLLIP, as key mediators of granule disassembly. In addition, we identify an autophagy-related pathway that promotes granule clearance. Collectively, this work establishes a general photoproximity labeling approach for unraveling intracellular protein interactomes and uncovers previously unexplored regulatory mechanisms of stress granule dynamics.

Proteasome resides in and dismantles plant heat stress granules constitutively

Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.

RNA damage compartmentalization by DHX9 stress granules

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.

Regulation of stress granule formation in human oligodendrocytes

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

Dysregulation of stress granule dynamics by DCTN1 deficiency exacerbates TDP-43 pathology in Drosophila models of ALS/FTD

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a major pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is aberrantly accumulated in the neurons of most patients with sporadic ALS/FTD and other TDP-43 proteinopathies, how TDP-43 forms cytoplasmic aggregates remains unknown. In this study, we show that a deficiency in DCTN1, a subunit of the microtubule-associated motor protein complex dynactin, perturbs the dynamics of stress granules and drives the formation of TDP-43 cytoplasmic aggregation in cultured cells, leading to the exacerbation of TDP-43 pathology and neurodegeneration in vivo. We demonstrated using a Drosophila model of ALS/FTD that genetic knockdown of DCTN1 accelerates the formation of ubiquitin-positive cytoplasmic inclusions of TDP-43. Knockdown of components of other microtubule-associated motor protein complexes, including dynein and kinesin, also increased the formation of TDP-43 inclusions, indicating that intracellular transport along microtubules plays a key role in TDP-43 pathology. Notably, DCTN1 knockdown delayed the disassembly of stress granules in stressed cells, leading to an increase in the formation of pathological cytoplasmic inclusions of TDP-43. Our results indicate that a deficiency in DCTN1, as well as disruption of intracellular transport along microtubules, is a modifier that drives the formation of TDP-43 pathology through the dysregulation of stress granule dynamics.

Optimizing TDP-43 silencing with siRNA-loaded polymeric nanovectors in neuronal cells for therapeutic applications: balancing knockdown and function.

TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA binding protein critical for regulating gene expression, including transcription, splicing, mRNA stability, and protein translation. Aggregation of pathological TDP-43 proteins in the cytoplasm of neurons and glial cells appears to be a common feature of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases such as frontotemporal dementia (FTD), contributing to motor neuron degeneration and clinical symptoms. Downregulation of TDP-43 expression to prevent or reduce the formation of pathological aggregates is a potential therapeutic approach for treating TDP-43-related diseases. However, therapeutic strategies to reduce TDP-43 aggregation face significant challenges, as the downregulation of TDP-43 must balance the need to maintain its normal functions, which are essential for RNA metabolism and cellular homeostasis. In this study, we developed novel polymeric nanovectors for the delivery of TDP-43 siRNAs in neuronal cells. These nanovectors were designed to provide adequate TDP-43 silencing to achieve the desired functional reduction of TDP-43 levels, thereby optimizing its impact on cellular functions. Our results demonstrate that the polymeric nanovector formulations effectively reduced TDP-43 mRNA and protein levels to an extent comparable to those observed with traditional lipid-based systems. Concurrently, the polymeric nanovectors exhibited an enhanced capacity to reduce stress granules (SG) formation and facilitate TDP-43-containing SG disassembly, while preserving its essential cellular functions. This study provides the first evidence that polymeric nanovectors may be a valuable tool for developing therapeutic strategies to treat TDP-43 protein diseases, such as ALS and FTD, by directly silencing TDP-43 to reduce its aggregation.

Alternative splicing of TaHSFA6e modulates heat shock protein-mediated translational regulation in response to heat stress in wheat.

Heat stress greatly threatens crop production. Plants have evolved multiple adaptive mechanisms, including alternative splicing, that allow them to withstand this stress. However, how alternative splicing contributes to heat stress responses in wheat (Triticum aestivum) is unclear. We reveal that the heat shock transcription factor gene TaHSFA6e is alternatively spliced in response to heat stress. TaHSFA6e generates two major functional transcripts: TaHSFA6e-II and TaHSFA6e-III. TaHSFA6e-III enhances the transcriptional activity of three downstream heat shock protein 70 (TaHSP70) genes to a greater extent than does TaHSFA6e-II. Further investigation reveals that the enhanced transcriptional activity of TaHSFA6e-III is due to a 14-amino acid peptide at its C-terminus, which arises from alternative splicing and is predicted to form an amphipathic helix. Results show that knockout of TaHSFA6e or TaHSP70s increases heat sensitivity in wheat. Moreover, TaHSP70s are localized in stress granule following exposure to heat stress and are involved in regulating stress granule disassembly and translation re-initiation upon stress relief. Polysome profiling analysis confirms that the translational efficiency of stress granule stored mRNAs is lower at the recovery stage in Tahsp70s mutants than in the wild types. Our finding provides insight into the molecular mechanisms by which alternative splicing improves the thermotolerance in wheat.

ALS-linked FUS R521C disrupts arginine methylation of UBAP2L and stress granule dynamics

Mutations in the fused-in-sarcoma (FUS) gene have been linked to familial amyotrophic lateral sclerosis (fALS). FUS aggregates in the cytosol and associates with stress granules (SGs) in pathological cases, whereas FUS is normally found in the nucleus. However, little is known about how FUS mutations cause neurodegeneration in ALS, which is distinguished by FUS-positive inclusion and stress granules. In this study, we investigated the mechanism of abnormal cytoplasmic aggregate formation caused by ALS-linked FUS mutations. FUS R521C interacted more with ubiquitin-associated protein 2-like (UBAP2L) and protein arginine methyltransferase 1 (PRMT1) than FUS WT, and PRMT1 and UBAP2L are sequestered into FUS R521C-positive stress granules under oxidative stress. PRMT1 asymmetrically demethylates UBAP2L, which is required for both SG assembly and disassembly. Furthermore, in FUS R521C, arginine methylation of UBAP2L is reduced, and the loss of PRMT1 increases FUS-positive SGs in oxidative stress. These results imply that an aberrant interaction between FUS-R521C and PRMT1 causes insufficient arginine methylation of UBAP2L, resulting in abnormal FUS-R521C-positive SGs remaining in the cytoplasm. This study could lead to the identification of a new pathogenic mechanism and therapeutic targets for the FUS mutation, which has been associated with abnormally increased protein interactions in ALS.Graphical abstract

Graphene Quantum Dots Modulate Stress Granule Assembly and Prevent Abnormal Phase Transition of Fused in Sarcoma Protein.

Protein liquid-liquid phase separation (LLPS) plays a crucial role in mediating dynamic assembly of different membraneless organelles such as stress granules (SGs). Dysregulation of dynamic protein LLPS leads to aberrant phase transition and amyloid aggregation which is closely associated with neurodegenerative diseases. In this study, we found that three types of graphene quantum dots (GQDs) exhibit potent activity in preventing SG formation and promoting SG disassembly. We next demonstrate that GQDs can directly interact with the SGs-containing protein fused in sarcoma (FUS), inhibit and reverse FUS LLPS, and prevent its abnormal phase transition. Moreover, GQDs display superior activity in preventing amyloid aggregation of FUS and disaggregating preformed FUS fibrils. Mechanistic study further demonstrates that GQDs with different edge-site exhibit distinct binding affinity to FUS monomers and fibrils, which accounts for their distinct activities in modulating FUS LLPS and fibrillation. Our work reveals the potent capability of GQDs in modulating SG assembly, protein LLPS, and fibrillation and sheds light on rational design of GQDs as effective modulators of protein LLPS for therapeutics application.

Sestrin2-mediated disassembly of stress granules dampens aerobic glycolysis to overcome glucose starvation

Sestrins are a small gene family of pleiotropic factors whose actions promote cell adaptation to a range of stress conditions. In this report we disclose the selective role of Sestrin2 (SESN2) in dampening aerobic glycolysis to adapt to limiting glucose conditions. Removal of glucose from hepatocellular carcinoma (HCC) cells inhibits glycolysis associated with the downregulation of the rate-limiting glycolytic enzyme hexokinase 2 (HK2). Moreover, the accompanying upregulation of SESN2 through an NRF2/ATF4-dependent mechanism plays a direct role in HK2 regulation by destabilizing HK2 mRNA. We show SESN2 competes with insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) for binding with the 3′-UTR region of HK2 mRNA. Interactions between IGF2BP3 and HK2 mRNA result in their coalescence into stress granules via liquid-liquid phase separation (LLPS), a process which serves to stabilize HK2 mRNA. Conversely, the enhanced expression and cytoplasmic localization of SESN2 under glucose deprivation conditions favors the downregulation of HK2 levels via decreases in the half-life of HK2 mRNA. The resulting dampening of glucose uptake and glycolytic flux inhibits cell proliferation and protect cells from glucose starvation-induced apoptotic cell death. Collectively, our findings reveal an intrinsic survival mechanism allowing cancer cells to overcome chronic glucose shortages, also providing new mechanistic insights into SESN2 as an RNA-binding protein with a role in reprogramming of cancer cell metabolism.

Abstract 4773: DEAD Box 1 (DDX1) protects its RNA targets in cells exposed to oxidative stress

Abstract DEAD box 1 (DDX1) belongs to the family of RNA helicases involved in all aspects of RNA metabolism, from transcription to transport, expression, and decay. DDX1 is overexpressed in a subset of retinoblastoma, neuroblastoma, and breast cancers. In breast cancer, the overexpression and cytoplasmic localization of DDX1 is correlated with poor prognosis. Cancer cells often exhibit high metabolic rates which can lead to genomic instability, oxidative stress, and endoplasmic stress. DDX1 is known to play a role in resolving genotoxic stress by participating in DNA double-strand break (DSB) repair. We propose that DDX1 also plays a key role as a stress response regulator in cells undergoing oxidative stress. When cells are exposed to oxidative stress, DDX1 is recruited to cytoplasmic stress granules (SGs). Although the exact function of SGs remains elusive, it is generally believed that tightly controlled SG assembly and disassembly are important for cell survival under stress. We found that while DDX1 is dispensable for SG assembly, depletion of DDX1 leads to impaired SG disassembly and hence, delayed stress recovery. Thus, DDX1 may play a role in stress resolution after the removal of stress conditions. To further understand the role of DDX1 in stress resolution, we performed RNA-immunoprecipitation followed by next-generation sequencing and identified DDX1-bound RNAs under normal and oxidative stress conditions. We found enhanced binding of DDX1 to its RNA targets in the presence of oxidative stress. Furthermore, we observed that DDX1 protects its target RNAs from degradation in the cytoplasm of cells undergoing oxidative stress. This RNA protection after stress exposure was more prominent in DDX1-overexpressing neuroblastoma cells, possibly explaining the faster stress recovery observed in these cells. Taken together, our results suggest that DDX1 plays a role in protecting cells from oxidative stress. This study may shed light on our understanding of cancer cell resistance to anticancer therapies. Citation Format: Mansi Garg, Roseline Godbout. DEAD Box 1 (DDX1) protects its RNA targets in cells exposed to oxidative stress. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4773.

Targeting the NEDP1 enzyme to ameliorate ALS phenotypes through stress granule disassembly.

The elimination of aberrant inclusions is regarded as a therapeutic approach in neurodegeneration. In amyotrophic lateral sclerosis (ALS), mutations in proteins found within cytoplasmic condensates called stress granules (SGs) are linked to the formation of pathological SGs, aberrant protein inclusions, and neuronal toxicity. We found that inhibition of NEDP1, the enzyme that processes/deconjugates the ubiquitin-like molecule NEDD8, promotes the disassembly of physiological and pathological SGs. Reduction in poly(ADP-ribose) polymerase1 activity through hyper-NEDDylation is a key mechanism for the observed phenotype. These effects are related to improved cell survival in human cells, and in C. elegans, nedp1 deletion ameliorates ALS phenotypes related to animal motility. Our studies reveal NEDP1 as potential therapeutic target for ALS, correlated to the disassembly of pathological SGs.

The Batten disease protein CLN3 is important for stress granules dynamics and translational activity

The assembly of membrane-less organelles such as stress granules (SGs) is emerging as central in helping cells rapidly respond and adapt to stress. Following stress sensing, the resulting global translational shutoff leads to the condensation of stalled mRNAs and proteins into SGs. By reorganizing cytoplasmic contents, SGs can modulate RNA translation, biochemical reactions, and signaling cascades to promote survival until the stress is resolved. While mechanisms for SG disassembly are not widely understood, the resolution of SGs is important for maintaining cell viability and protein homeostasis. Mutations that lead to persistent or aberrant SGs are increasingly associated with neuropathology and a hallmark of several neurodegenerative diseases. Mutations in CLN3 are causative of juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease affecting children also known as Batten disease. CLN3 encodes a transmembrane lysosomal protein implicated in autophagy, endosomal trafficking, metabolism, and response to oxidative stress. Using a HeLa cell model lacking CLN3, we now show that CLN3KO is associated with an altered metabolic profile, reduced global translation, and altered stress signaling. Furthermore, loss of CLN3 function results in perturbations in SG dynamics, resulting in assembly and disassembly defects, and altered expression of the key SG nucleating factor G3BP1. With a growing interest in SG-modulating drugs for the treatment of neurodegenerative diseases, novel insights into the molecular basis of CLN3 Batten disease may reveal avenues for disease-modifying treatments for this debilitating childhood disease.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress.

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

Pab1 acetylation at K131 decreases stress granule formation in Saccharomyces cerevisiae

Under environmental stress, such as glucose deprivation, cells form stress granules-the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative diseases, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. An important aspect of stress granule formation is the presence of posttranslational modifications on core proteins. One of those modifications is lysine acetylation, which is regulated by either a lysine acetyltransferase or a lysine deacetylase enzyme. This work deciphers the impact of lysine acetylation on an essential protein found in Saccharomyces cerevisiae stress granules, poly(A)-binding protein (Pab1). We demonstrated that an acetylation mimic of the lysine residue in position 131 reduces stress granule formation upon glucose deprivation and other stressors such as ethanol, raffinose, and vanillin. We present genetic evidence that the enzyme Rpd3 is the primary candidate for the deacetylation of Pab1-K131. Further, our electromobility shift assay studies suggest that the acetylation of Pab1-K131 negatively impacts poly(A) RNA binding. Due to the conserved nature of stress granules, therapeutics targeting the activity of lysine acetyltransferases and lysine deacetylase enzymes may be a promising route to modulate stress granule dynamics in the disease state.

Amyotrophic Lateral Sclerosis, FUS and Protein Synthesis Defects.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects the motor system. It is a very heterogeneous disorder, so far more than 40 genes have been described as responsible for ALS. The cause of motor neuron degeneration is not yet fully understood, but there is consensus in the literature that it is the result of a complex interplay of several pathogenic processes, which include alterations in nucleocytoplasmic transport, defects in transcription and splicing, altered formation and/or disassembly of stress granules and impaired proteostasis. These defects result in protein aggregation, impaired DNA repair, mitochondrial dysfunction and oxidative stress, neuroinflammation, impaired axonal transport, impaired vesicular transport, excitotoxicity, as well as impaired calcium influx. We argue here that all the above functions ultimately lead to defects in protein synthesis. Fused in Sarcoma (FUS) is one of the genes associated with ALS. It causes ALS type 6 when mutated and is found mislocalized to the cytoplasm in the motor neurons of sporadic ALS patients (without FUS mutations). In addition, FUS plays a role in all cellular functions that are impaired in degenerating motor neurons. Moreover, ALS patients with FUS mutations present the first symptoms significantly earlier than in other forms of the disease. Therefore, the aim of this review is to further discuss ALS6, detail the cellular functions of FUS, and suggest that the localization of FUS, as well as protein synthesis rates, could be hallmarks of the ALS phenotype and thus good therapeutic targets.

Role of Proteostasis Regulation in the Turnover of Stress Granules

RNA-binding proteins (RBPs) and RNAs can form dynamic, liquid droplet-like cytoplasmic condensates, known as stress granules (SGs), in response to a variety of cellular stresses. This process is driven by liquid-liquid phase separation, mediated by multivalent interactions between RBPs and RNAs. The formation of SGs allows a temporary suspension of certain cellular activities such as translation of unnecessary proteins. Meanwhile, non-translating mRNAs may also be sequestered and stalled. Upon stress removal, SGs are disassembled to resume the suspended biological processes and restore the normal cell functions. Prolonged stress and disease-causal mutations in SG-associated RBPs can cause the formation of aberrant SGs and/or impair SG disassembly, consequently raising the risk of pathological protein aggregation. The machinery maintaining protein homeostasis (proteostasis) includes molecular chaperones and co-chaperones, the ubiquitin-proteasome system, autophagy, and other components, and participates in the regulation of SG metabolism. Recently, proteostasis has been identified as a major regulator of SG turnover. Here, we summarize new findings on the specific functions of the proteostasis machinery in regulating SG disassembly and clearance, discuss the pathological and clinical implications of SG turnover in neurodegenerative disorders, and point to the unresolved issues that warrant future exploration.