The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Stress Granule Core Protein-Derived Peptides Inhibit Assembly of Stress Granules and Improve Sorafenib Sensitivity in Cancer Cells.

Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.

TRIM25 predominately associates with anti-viral stress granules.

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

Altering biomolecular condensates as a potential mechanism that mediates cannabidiol effect on glioblastoma.

Glioblastoma (GBM) is an extremely aggressive primary brain tumor with poor prognosis, short survival time post-diagnosis and high recurrence. Currently, no cure for GBM exists. The identification of an effective therapeutic modality for GBM remains a high priority amongst medical professionals and researches. In recent studies, inhalant cannabidiol (CBD) has demonstrated promise in effectively inhibiting GBM tumor growth. However, exactly how CBD treatment affects the physiology of these tumor cells remains unclear. Stress granules (SG) (a sub-class of biomolecular condensates (BMC)) are dynamic, membrane-less intracellular microstructures which contain proteins and nucleic acids. The formation and signaling of SGs and BMCs plays a significant role in regulating malignancies. This study investigates whether inhaled CBD may play an intervening role towards SGs in GBM tumor cells. Integrated bioinformatics approaches were preformed to gain further insights. This includes use of Immunohistochemistry and flow cytometry to measure SGs, as well as expression and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The findings of this study reveal that CBD receptors (and co-regulated genes) have the potential to play an important biological role in the formation of BMCs within GBM. In this experiment, CBD treatment significantly increased the volume of TIAR-1. This increase directly correlated with elevation in both eIF2α expression and p-eIF2α in CBD treated tissues in comparison to the placebo group (p < 0.05). These results suggest that inhalant CBD significantly up-regulated SGs in GBM, and thus support a theory of targeting BMCs as a potential therapeutic substrate for treating GBM.

DDX6 modulates P-body and stress granule assembly, composition, and docking.

Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress granules form in part by intermolecular RNA-RNA interactions and can be limited by components of the RNA chaperone network, which inhibits RNA-driven aggregation. Herein, we demonstrate that the DEAD-box helicase DDX6, a P-body component, can also limit the formation of stress granules, independent of the formation of P-bodies. In an ATPase, RNA-binding dependent manner, DDX6 limits the partitioning of itself and other RNPs into stress granules. When P-bodies are limited, proteins that normally partition between stress granules and P-bodies show increased accumulation within stress granules. Moreover, we show that loss of DDX6, 4E-T, and DCP1A increases P-body docking with stress granules, which depends on CNOT1 and PAT1B. Taken together, these observations identify a new role for DDX6 in limiting stress granules and demonstrate that P-body components can influence stress granule composition and docking with P-bodies.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) promotes stress granule formation via YBX1 phosphorylation in ovarian cancer.

APE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.

Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in KrasG12D-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.

Escherichia coli-Induced cGLIS3-Mediated Stress Granules Activate the NF-κB Pathway to Promote Intrahepatic Cholangiocarcinoma Progression.

Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.

Formation of the NLRP3 inflammasome inhibits stress granule assembly by multiple mechanisms.

Proper regulation of cellular response to environmental stress is crucial for maintaining biological homeostasis and is achieved by the balance between cell death processes, such as the formation of the pyroptosis-inducing NLRP3 inflammasome, and pro-survival processes, such as stress granule (SG) assembly. However, the functional interplay between these two stress-responsive organelles remains elusive. Here, we identified DHX33, a viral RNA sensor for the NLRP3 inflammasome, as a SG component, and the SG-nucleating protein G3BP as an NLRP3 inflammasome component. We also found that a decrease in intracellular potassium (K+) concentration, a key 'common' step in NLRP3 inflammasome activation, markedly inhibited SG assembly. Therefore, when macrophages are exposed to stress stimuli with the potential to induce both SGs and the NLRP3 inflammasome, such as cytoplasmic poly(I:C) stimulation, they preferentially form the NLRP3 inflammasome but avoid SG assembly by sequestering G3BP into the inflammasome and by inducing a reduction in intracellular K+ levels. Thus, under such conditions, DHX33 is primarily utilized as a viral RNA sensor for the inflammasome. Our data reveal the functional crosstalk between NLRP3 inflammasome-mediated pyroptosis and SG-mediated cell survival pathways and delineate a molecular mechanism that regulates cell-fate decisions and anti-viral innate immunity under stress.

HuR-positive stress granules: Potential targets for age-related osteoporosis.

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target ß-catenin by binding and recruiting ß-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of ß-catenin into HuR-positive SGs, facilitated ß-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

YBX1 Underwent Phase Separation into Stress Granules Stimulated by Ionizing Radiation.

Stress granules (SGs) are formed through liquid-liquid phase separation (LLPS), in response to external stimuli. YBX1, an integral component of SGs, plays a crucial role in tumor progression and cellular stress response. This study aims to elucidate the mechanisms and specific biological implications of YBX1 in SG formation, along with the identification of key regions and interacting proteins. Our observations indicate that YBX1 rapidly undergoes liquid-liquid phase separation, leading to SG formation in response to 8 Gy X-ray irradiation within 1 h, with SGs reverting to their original state after 5 h. There was a potential interaction between ATXN2L and YBX1, persisting YBX1 within the SGs. Our data suggested a potential interaction between ATXN2L and YBX1, and it remained associated with YBX1 within the SGs. Furthermore, our subsequent studies demonstrate that targeting ATXN2L can diminish the recruitment of YBX1 to stress granules (SGs), consequently enhancing the radiosensitivity of HeLa cells.

Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma Cells.

PURPOSE: Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive. METHODS AND MATERIALS: The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells. RESULTS: Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells. CONCLUSIONS: SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress.

A novel stroke rehabilitation strategy and underlying stress granule regulations through inhibition of NLRP3 inflammasome activation.

OBJECTIVE: Dynamic changes in ischemic pathology after stroke suggested a "critical window" of enhanced neuroplasticity immediately after stroke onset. Although physical exercise has long been considered a promising strategy of stroke rehabilitation, very early physical exercise may exacerbate brain injury. Since remote ischemic conditioning (RIC) promotes neuroprotection and neuroplasticity, the present study combined RIC with sequential exercise to establish a new rehabilitation strategy for a better rehabilitative outcome. METHODS: A total of 120 adult male Sprague-Dawley rats were used and divided into five groups: (1) sham, (2) stroke, (3) stroke with exercise, (4) stroke with RIC, and (5) stroke with RIC followed by exercise. Brain damage was evaluated by infarct volume, neurological deficit, cell death, and lactate dehydrogenase (LDH) activity. Long-term functional outcomes were determined by grid walk tests, rotarod tests, beam balance tests, forelimb placing tests, and the Morris water maze. Neuroplasticity was evaluated through measurements of both mRNA and protein levels of synaptogenesis (synaptophysin [SYN], post-synaptic density protein-95 [PSD-95], and brain-derived neurotrophic factor [BDNF]) and angiogenesis (vascular endothelial growth factor [VEGF], angiopoietin-1 [Ang-1], and angiopoietin-2 [Ang-2]). Inflammasome activation was measured by concentrations of interleukin-18 (IL-18) and IL-1ß detected by enzyme-linked immunosorbent assay (ELISA) kits, mRNA expressions of NLR pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), IL-18 and IL-1ß, and protein quantities of NLRP3, ASC, cleaved-caspase-1, gasdermin D-N (GSDMD-N), and IL-18 and IL-1ß. Stress granules (SGs), including GTPase-activating protein-binding protein 1 (G3BP1), T cell-restricted intracellular antigen-1 (TIA1), and DEAD-box RNA helicase 3X (DDX3X) were evaluated at mRNA and protein levels. The interactions between DDX3X with NLRP3 or G3BP1 were determined by immunofluorescence and co-immunoprecipitation. RESULTS: Early RIC decreased infarct volumes, neurological deficits, cell death, and LDH activity at post-stroke Day 3 (p < 0.05). All treatment groups showed significant improvement in functional outcomes, including sensory, motor, and cognitive functions. RIC and exercise, as compared to RIC or physical exercise alone, had improved functional outcomes after stroke (p < 0.05), as well as synaptogenesis and angiogenesis (p < 0.05). RIC significantly reduced mRNA and protein expressions of NLRP3 (p < 0.05). SGs formation peaked at 0 h after ischemia, then progressively decreased until 24 h postreperfusion, which was reversed by RIC (p < 0.05). The assembly of SGs consumed DDX3X and then inhibited NLRP3 inflammasome activation. CONCLUSIONS: RIC followed by exercise induced a better rehabilitation in ischemic rats, while early RIC alleviated ischemia-reperfusion injury via stress-granule-mediated inhibition of NLRP3 inflammasome.

An emerging role for the endoplasmic reticulum in stress granule biogenesis.

Stress granules (SGs), structurally dynamic, optically resolvable, macromolecular assemblies of mRNAs, RNA binding proteins (RBPs), translation factors, ribosomal subunits, as well as other interacting proteins, assemble in response to cell stress conditions that elicit phosphorylation of eukaryotic initiation factor 2α (eIF2α) and consequently, the inactivation of translation initiation. SG biology is conserved throughout eukaryotes and has recently been linked to the pathological sequelae of neurodegenerative disorders, cancer biology, and viral infection. Substantial insights into mechanisms of SG biogenesis, and more broadly the phenomenon of biological liquid-liquid phase separation (LLPS), have been aided by detailed proteomic and transcriptomic studies as well as in vitro reconstitution approaches. A particularly interesting and largely unexplored element of SG biology is the cell biological context of SG biogenesis, including its subcellular organization and more recently, evidence that the endoplasmic reticulum (ER) membrane may serve important functions in RNA granule biology generally and SG biogenesis specifically. A central role for the ER in SG biogenesis is discussed and a hypothesis linking SG formation on the ER to the trafficking, localization and de novo translation of newly exported mRNAs is presented.

Inhibition of cGAS-STING pathway by stress granules after activation of M2 macrophages by human mesenchymal stem cells against drug induced liver injury.

OBJECTIVE: Cells can produce stress granules (SGs) to protect itself from damage under stress. The cGAS-STING pathway is one of the important pattern recognition pathways in the natural immune system. This study was investigated whether human mesenchymal stem cells (hMSCs) could protect the liver by inducing M2 macrophages to produce SGs during acute drug induced liver injury (DILI) induced by acetaminophen (APAP). METHODS: After intragastric administration of APAP in vivo to induce DILI mice model, hMSCs were injected into the tail vein. The co-culture system of hMSCs and M2 macrophages was established in vitro. It was further use SGs inhibitor anisomicin to intervene M2 macrophages. The liver histopathology, liver function, reactive oxygen species (ROS) level, apoptosis pathway, endoplasmic reticulum stress (ERS) level, SGs markers (G3BP1/TIA-1), cGAS-STING pathway, TNF-α, IL-6, IL-1ß mRNA levels in liver tissue and M2 macrophages were observed. RESULTS: In vivo experiments, it showed that hMSCs could alleviate liver injury, inhibite the level of ROS, apoptosis and ERS, protect liver function in DILI mice. The mount of M2 was increased in the liver. hMSCs could also induce the production of SGs, inhibit the cGAS-STING pathway and reduce TNF-α, IL-6, IL-1ß mRNA expression. The results in vitro showed that hMSCs could induce the production of SGs in macrophages, inhibit the cGAS-STING pathway, promote the secretion of IL-4 and IL-13, and reduce TNF-α, IL-6, IL-1ß mRNA level in cells. In the process of IL-4 inducing M2 macrophage activation, anisomycin could inhibit the production of SGs, activate the cGAS-STING pathway, and promote the inflammatory factor TNF-α, IL-6, IL-1ß mRNA expression in cells. CONCLUSIONS: HMSCs had a protective effect on acute DILI in mice induced by APAP. Its mechanism might involve in activating M2 type macrophages, promoting the production of SGs, inhibiting the cGAS-STING pathway, and reducing the expression of pro-inflammatory factors in macrophages, to reduce hepatocytes damage.

Stress granules plug and stabilize damaged endolysosomal membranes.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

Role of stress granules in tumorigenesis and cancer therapy.

Stress granules (SGs) are membrane-less organelles that cell forms via liquid-liquid phase separation (LLPS) under stress conditions such as oxidative stress, ER stress, heat shock and hypoxia. SG assembly is a stress-responsive mechanism by regulating gene expression and cellular signaling pathways. Cancer cells face various stress conditions in tumor microenvironment during tumorigenesis, while SGs contribute to hallmarks of cancer including proliferation, invasion, migration, avoiding apoptosis, metabolism reprogramming and immune evasion. Here, we review the connection between SGs and cancer development, the limitation of SGs on current cancer therapy and promising cancer therapeutic strategies targeting SGs in the future.

LIMK1 m6A-RNA methylation recognized by YTHDC2 induces 5-FU chemoresistance in colorectal cancer via endoplasmic reticulum stress and stress granule formation.

LIM kinase 1 (LIMK1) is a member of the LIMK family that has been considered to be involved in chemoresistance in various tumors, and N6-methyladenosine (m6A) is the most abundant nucleotide modification on mRNA. However, whether elevated expression of LIMK1 leads to chemoresistance due to m6A modification remains to be further studied. The findings of our study indicate that high LIMK1 expression in colorectal cancer (CRC) cells promotes cell proliferation and increases resistance to 5-fluorouracil (5-FU). Moreover, downregulation of YTH domain-containing 2 (YTHDC2), an m6A "reader", in CRC cells resulted in decreased recognition and binding to the m6A site "GGACA" in LIMK1 mRNA, thereby increasing LIMK1 mRNA stability and expression. Furthermore, the overexpression of LIMK1 facilitated eIF2α phosphorylation, which induced endoplasmic reticulum (ER) stress and promoted stress granule (SG) formation, ultimately leading to 5-FU resistance. This study evaluated the specificity of the YTHDC2/LIMK1/eIF2α signalling axis and the efficacy of related drugs in modulating 5-FU sensitivity in CRC.