Journal of Molecular Cell Biology

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

Mitophagy is generally considered to promote cell survival by removing damaged mitochondria in response to mitochondrial stress, whereas apoptosis occurs during prolonged stress. However, the mechanisms that determine cell survival and cell death under these stress conditions remain poorly understood. Here, we showed that cytoplasmic mRNA granules, designated as mitophagy-induced mRNA granules (mitoRGs), were formed transiently and played an important role in cell fate decisions during PINK1/Parkin-dependent mitophagy. Although some components, such as G3BP1, were shared with stress granules (SGs), mitoRGs were distinct from SGs because mitoRG assembly required the mitochondrial protein phosphatase PGAM5. In response to mitochondrial stress, PGAM5 was released into the cytosol from mitochondria and incorporated into mitoRGs, but was then released back into the cytosol during mitoRG disassembly following prolonged mitochondrial stress, corresponding with the induction of apoptosis. Impairment of mitoRG assembly through G3BP1 depletion sensitized cells to apoptosis during mitophagy in a PGAM5-dependent manner. These results suggest that mitoRGs regulate cell fate decisions by spatiotemporally controlling PGAM5 and its pro-apoptotic activity during PINK1/Parkin mitophagy.

Tumor heterogeneity highlights the necessity of precision cancer medicine, making the evaluation and screening of anticancer drugs a core challenge in cancer therapy. However, current cell-based efficacy assessment methods struggle to quantify the holistic impact of drugs on cellular behavior through specific target engagement. Here, we proposed a novel approach (DL-TCP-FRET) that integrates phenotypic and target-related evaluations: the logistic fitting analysis is performed on time- and concentration-dependent cellular phenotypic characteristics to construct a phenotypic score (P), while a target score (T) is established based on the FRET efficiency between target proteins. These two scores were then further combined to generate a unified drug efficacy score (PT). Validation in A549 cells demonstrated that our method can reliably distinguish EGFR-TKIs from non-targeted drugs. DL-TCP-FRET simplifies the experimental workflow of drug efficacy evaluation and improves the accuracy of targeted drug identification, providing a novel strategy for advancing precision cancer therapy.

In eukaryotic cells, genomic DNA is packaged into chromatin with nucleosomes formed by core histones H2A, H2B, H3, and H4, and further stabilized by the linker histone H1. During the early stages of retroviral infection, such as with murine leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), host core and H1 histones are rapidly deposited onto unintegrated viral DNAs upon nuclear entry. These unintegrated viral DNAs are transcriptionally silenced through histone post-translational modifications (PTMs), including high levels of H3K9 trimethylation and low levels of H3 acetylation. Linker histone H1 is closely associated with chromatin compaction and histone PTMs, suggesting a potential role in regulating retroviral DNA fate. In this study, we demonstrate that simultaneous knockdown of four somatic H1 variants (H1.2, H1.3, H1.4, and H1.5) in K562 cells reverses the silencing of unintegrated HIV-1 DNA, resulting in increased viral expression. Notably, this effect was specific to HIV-1, as the same H1 depletion did not alter the silencing of MLV unintegrated DNA. These results reveal distinct roles of H1 in regulating HIV-1 and MLV unintegrated DNA expression.

Summary/AbstractHistone methylation is a dynamic and reversible epigenetic modification that critically controls the progression of human diseases, including infections and cancers. Here we reported that histone lysine demethylases (KDMs) in the KDM5 family KDM5A/B play profound roles in suppressing lytic reactivation of oncogenic human herpesvirus 8 (HHV-8), i.e., Kaposis sarcoma-associated herpesvirus (KSHV), as well as antiviral/antitumor innate immune responses in KSHV-infected B-cell lymphomas. We showed that KSHV lytic replication decreases KDM5A/B protein stability by enhancing their K-48 linked polyubiquitination while KDM5A/B depletion facilitates KSHV lytic reactivation. Mechanistic studies illustrated that KDM5A/B associate with KSHV LANA protein and dampen its chromatin association at both KSHV viral lytic promoter and promoters of antitumor immune-responsive genes (IRGs). In comparisons to normal B cells, KDM5A/B expression significantly increased in B-cell lymphoma cells, including KSHV-positive primary effusion lymphoma (PEL). We demonstrated that KDM5A/B inhibition remarkably induces both KSHV lytic reactivation and innate immune responses in PEL cells, resulting in a strong viral oncolytic effect, both in vitro in cell cultures and in vivo using a PEL xenograft mouse model. Overall, our studies identified the novel functions of KDM5A/B to silence KSHV lytic replication and antiviral/antitumor innate immune responses, which can be blocked to benefit the treatment of KSHV-associated B-cell lymphomas that are usually aggressive and difficult to treat.

During viral infection, viral replication perturbs endoplasmic reticulum (ER) homeostasis and triggers the unfolded protein response (UPR). XBP1s, a transcription factor generated by one branch of the UPR, is known to potentiate both innate and adaptive immunity, but its role in antiviral responses remains incompletely understood beyond its ability to augment type I interferon (IFN) mRNA induction. Here, we show that XBP1s positively regulates the RIG-I-like receptors (RLRs), ribonuclease L (RNase L), and protein kinase R (PKR) pathways, indicating that it enhances all three major antiviral response pathways. We further show that RNase L activation rapidly decreases XBP1 mRNA levels in an RNase activity-dependent manner, leading to a prompt reduction in XBP1s expression. Consistent with this, RNase L deletion significantly increased both thapsigargin-mediated XBP1s induction and XBP1s expression following Japan encephalitis virus infection. Poly(I:C)-induced IFNB mRNA expression was significantly enhanced in RNase L-knockout cells. This enhancement was completely abolished by RNase L reconstitution. XBP1 knockdown also significantly attenuated IFNB mRNA expression in RNase L-knockout cells. These findings suggest a negative-feedback loop in which RNase L suppresses XBP1s, thereby fine-tuning antiviral responsiveness during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/713401v1_ufig1.gif" ALT="Figure 1000"> View larger version (19K): org.highwire.dtl.DTLVardef@112d312org.highwire.dtl.DTLVardef@df79a9org.highwire.dtl.DTLVardef@1ac571borg.highwire.dtl.DTLVardef@18ac610_HPS_FORMAT_FIGEXP M_FIG C_FIG

The cell cycle comprises different phases and is a tightly regulated process at the molecular level. During the cell cycle, two key events occurred: DNA duplication during the S phase and chromosome segregation during mitosis. Accurate cell cycle progression, achieved through faithful chromosome segregation, is essential for maintaining cell fidelity. Long noncoding RNAs are a subclass of noncoding RNA that are longer than 200 bp and form RNA protein complexes (RNPs) to regulate various biological processes. Herein, we demonstrate that lncRNA NORM is involved in regulating the cell cycle by maintaining proper chromosome segregation. NORM exhibited G2 phase-specific expression, and the depletion of NORM resulted in a significant G2/M arrest. NORM-depleted cells failed to progress in mitosis and showed defects in chromosome segregation. We further demonstrated that NORM binds to proteins such as Plk1 and Nsun2. Depletion of NORM hindered the interaction between Plk1 and Bub1, resulting in reduced kinetochore localization of Plk1 during prometaphase. Our results also show that the depletion of NORM affects the binding of Nsun2 protein to CDK1 mRNA and, consequently, the stabilization of CDK1 at the protein level. Altogether, our results demonstrate that NORM regulates chromosome segregation by mediating the interaction between Plk1 and Bub1.

ATAD2 possesses a C-terminal bromodomain (BRD) that plays a critical role in recognizing and binding to acetylated lysine residues. However, because the native intracellular structure of ATAD2 remains poorly defined, the mechanisms by which the ATAD2 BRD recruits acetylated histones and the regulatory pathways involved are not yet understood. In this study, we report that the ATAD2 BRD mediates the formation of liquid-liquid phase separation (LLPS) of ATAD2 in cells. This phase separation promotes the process of histone H4 acetylation, leading to the up-regulation of C-MYC, CCND3, and ATF2 gene expression and the facilitation of chromatin remodeling. Our findings elucidate a vital function of ATAD2, wherein BRD-mediated LLPS drives histone acetylation to promote cellular chromatin remodeling.

Vascular plasticity is a crucial biological asset enabling our bodies to rapidly adapt to infections and acute inflammation. However, repeated insult during chronic disease can result in these vascular adaptations becoming irreversible, thereby driving disease progression and fibrosis. This study aimed to understand if phenotypic changes in endothelial cell (EC) identity could be indicative of progressive fibrosis and thereby offer new diagnostic and therapeutic opportunities for patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Previous research has documented that a significant shift in EC transcriptomic signature occurs during liver fibrosis in both pre-clinical models and patients. However, the protein expression profile, phenotype and functional role of these new EC subpopulations that are induced during fibrogenesis is unclear. In this study, we integrate high-resolution imaging, proteomic and transcriptomic analysis which collectively highlight a central role for endothelial-to-mesenchymal transition (EndMT)-induced EC plasticity in the derivation of fibrosis-associated EC (FAEC). We demonstrate that: 1) full spectrum flow cytometry can provide new opportunities to categorize and phenotype EC subpopulations, 2) two distinct EndMT-derived FAEC subpopulations expand during fibrogenesis; THY1.2+ICAM1+ and TAGLN+MCAM+ EC that display unique immunomodulatory and metabolic phenotypes, 3) TAGLN+ FAEC are a conserved, pro-fibrotic cell type arising at early stages of MASLD, and 4) increased hepatic expression of TAGLN is significantly associated with detrimental patient outcomes at all stages of liver disease. This study will pave the way for the development of FAEC-specific diagnostic and therapeutic approaches to tackle progressive fibrotic disease.

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

Circular RNAs (circRNAs) originate from backsplicing of numerous genes in animals, but the functions of most circRNAs remain elusive. We previously demonstrated that circZNF827 forms a complex with hnRNPL/K and its host gene-encoded protein ZNF827 that acts in the nucleus to transcriptionally repress the nerve growth factor receptor (NGFR/p75NTR) gene during neuronal differentiation (Hollensen, 2020) [1]. To explore the mechanism of action, and to assess a potential role of the circZNF827-hnRNP complex on additional loci, we scrutinized the genome-wide consequences of circZNF827 and/or hnRNPL knockdown at the transcriptomic and epigenetic level. RNA-sequencing and CUT&RUN confirmed that NGFR and additional loci are transcriptionally repressed by the circZNF827-protein complex, and that these are primarily enriched for H3K27me3 signatures. Only a fraction of the massive transcriptomic changes could be ascribed to a direct circZNF827 transcription-regulated phenotype, suggesting that initial key regulatory events elicited by the circZNF827-hnRNP complex likely lead to a secondary response, which further augments neuronal differentiation.

Varicella Zoster Virus (VZV) is a dsDNA virus that infects dermal cells and causes characteristic cutaneous lesions. The virus undergoes neurotropism and later causes secondary cycles of infection. In the host nucleus, Promyelocytic Leukaemia Nuclear Bodies (PML-NBs) spontaneously form around the VZV genome to repress viral gene expression. VZV encodes for a ubiquitin E3 ligase ORF61 to disperse PML-NBs and alleviate repression. ORF61 functions as a ubiquitin E3 ligase with a conserved RING domain at the N-terminal end. It carries three SUMO-interacting motifs (SIMs) that mediate interactions with SUMOylated proteins within PML bodies. The mechanism by which ORF61 disperses PML-NBs is poorly understood. To understand how ORF61 interacts with SUMOylated proteins, we investigated its interaction with SUMO and studied its SUMO-Targeted Ubiquitin Ligase (STUbL) activity. Our studies reveal that ORF61 co-opts the E2D family for ubiquitination activity. A specific network of interactions between the E2 enzyme, ORF61, and Ub facilitates polyubiquitination. ORF61 can synthesize branched polyubiquitin chains of K11, K48, and K63 linkages. The C-terminal SIM in ORF61 is a high-affinity binder of SUMO chains. Utilizing the SIM, ORF61 targets specific lysines on SUMO chains for ubiquitination. These studies provide crucial insights into the functional mechanism of viral STUbL ORF61.

Hepatitis B and D virus (HBV, HDV) enter hepatocytes through a coordinated process mediated by a receptor complex consisting of sodium taurocholate co transporting polypeptide (NTCP) and its entry cofactors, including epidermal growth factor receptor (EGFR). Here, we established an in vitro assay to evaluate the NTCP-EGFR interaction and identified Oxy229, an oxysterol-based compound that disrupted this molecular interaction. Oxy229 selectively inhibited HBV and HDV infection to HepG2-NTCP cells and primary human hepatocytes. Mechanistic analysis revealed that Oxy229 impaired the relocalization of the HBV-NTCP complex from plasma membrane to intracellular vesicles. Notably, Oxy229 did not compromise the physiological functions of NTCP and EGFR, i.e., bile acid transport and activation of downstream EGFR signaling pathways including Ras-MAPK and PI3K-Akt pathways, indicating selective inhibition of viral entry. Compound derivative analysis identified Oxy283, which acquired dual inhibitory activity against both NTCP-EGFR interaction and NTCP multimerization, resulting in enhanced anti-HBV potency. These findings establish the functional significance of the NTCP-receptor complex formation in HBV/HDV entry and highlight this machinery as a potential target for antiviral intervention.

Mesenchymal stem cells (MSCs) have garnered significant attention over the past three decades due to their robust regenerative potential, primarily mediated by their paracrine activity by releasing soluble bioactive factors and extracellular vesicles (EVs). The MSC secretome plays a pivotal role in wound healing by influencing cellular migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. SPARC (Secreted Protein Acidic and Rich in Cysteine), a multifunctional ECM glycoprotein involved in tissue repair and remodeling, regulates key processes such as cell migration, proliferation, angiogenesis, and survival. Despite its known role in ECM dynamics, the impact of SPARC expression on the regenerative properties of MSCs remains underexplored. In this study, we hypothesized that SPARC overexpression in MSCs enhances their secretomes regenerative capacity. Using lentiviral systems, we generated SPARC-overexpressing (+SPARC) and SPARC-knockdown (KD-SPARC) MSCs to investigate SPARCs role in wound healing. Conditioned media (CM) derived from these MSCs were analyzed in vitro for their effects on human skin keratinocytes and fibroblasts. Our results revealed that SPARC expression significantly influences cell-specific migration and cell cycle. Furthermore, in an in vivo wound healing model, CM from +SPARC MSCs accelerated regeneration, while SPARC absence in MSCs CM delayed the healing process. These findings underscore the critical role of SPARC in modulating MSC secretome composition and enhancing its regenerative efficacy. This study highlights SPARC as a promising therapeutic target for the development of advanced regenerative therapies aimed at improving cutaneous wound healing outcomes.

Presenilin 1 (PS1), a key pathogenic factor in familial Alzheimers disease, is implicated in regulation of mitochondrial functions, yet its precise sub-mitochondrial localization and underlying mechanisms remain poorly understood. In this study, we generated PS1 knockout (PS1 KO) cell lines to investigate the role of PS1 in mitochondrial structure and function. Our results demonstrated that PS1 is directly localized to the mitochondrial inner membrane. PS1 deficiency led to reduced ATP production, impaired mitochondrial respiration capacity, decreased mitochondrial membrane potential, disrupted Ca2+ homeostasis, and elevated reactive oxygen species (ROS) accumulation. Moreover, loss of PS1 caused abnormal mitochondrial cristae structure. Further analysis revealed that PS1 interacts with mitochondrial inner membrane proteins. Its absence promotes ATAD3A oligomerization and disrupts its arrangement at mitochondrial cristae junctions, leading to expansion of the mitochondria-associated membrane (MAM) and instability of mitochondrial DNA (mtDNA). Our findings demonstrate that PS1 acts as a central regulator of mitochondrial cristae morphogenesis by modulating protein interaction networks at cristae junctions, thereby illuminating fundamental molecular mechanisms contributing to mitochondrial dysfunctions in Alzheimers disease.

Yin Yang 1 (YY1) is a multifunctional transcription factor and mammalian Polycomb Group (PcG) protein critical for lymphocyte development. While YY1 is essential for early T-cell development and survival, the underlying epigenetic mechanisms by which YY1 regulates early T-cell development are not fully understood. Herein, we utilized the YY1 PcG function conditional knockout mouse model (Yy1-/{Delta}REPO) by CRISPR/Cas9 to further dissect the underlying mechanisms. Yy1-/{Delta}REPO mice show early T cell development blockage at the double-negative (DN) 3 to single positive T cell transition with expansion of the DN3 population. Yy1-/{Delta}REPO DN3 cells are highly proliferative, but more prone to apoptosis, leading to reduced single positive T cells output. The genetic network governing T cell differentiation is deregulated in Yy1-/{Delta}REPO DN3 T cells. The YY1 REPO deletion leads to downregulation of DNA demethylase enzyme Tet1 and Tet2 expressions in DN3 cells with no change of Tet3. Pharmacologic inhibition of TET catalytic activity blocked DN-to-DP progression at the DN3 stage, whereas re-expression of TET2 catalytic domain in Yy1-/{Delta}REPO DN thymocytes partially rescued T cell differentiation. Together, our study demonstrates that YY1-mediated PcG function is essential for the DN3 to SP T cell transition and YY1-TET2 axis promotes proper DN3 differentiation.

Liver sinusoidal endothelial cells (LSECs) play essential roles in liver regeneration after injury, but the underlying mechanisms remain incompletely defined. Here we report that leukemia inhibitory factor (LIF), which is rapidly induced after liver injury, acts as a key regulator of LSECs-driven liver regeneration through interaction with LSECs-enriched LIF receptor (LIFR). LIF directly stimulates LSECs proliferation and induces hepatocyte growth factor (HGF) release in a dose-dependent manner via LIFR signaling in LSECs, thereby indirectly promoting hepatocyte proliferation. Systemic LIF neutralization or endothelial cells (ECs)-specific Lifr loss impairs liver regeneration, whereas low-titer AAV-mediated LIF expression increases vascular density, elevates circulating HGF, and improves early liver recovery after partial hepatectomy (PHx) in mice. Together, these findings establish LIF-LIFR as a previously unrecognized endothelial axis to promote hepatocyte proliferation and suggest potential therapeutic strategies to enhance liver repair in patients. HighlightsO_LILIF is upregulated after liver injury and LIF neutralization impairs liver recovery. C_LIO_LILIFR displays the highest expression in ECs; endothelial-specific Lifr deletion delays liver regeneration after injury. C_LIO_LILIF mediates a positive feedback loop including LSECs proliferation as well as HGF release via LIFR pathway. C_LIO_LILIF overexpression increases liver-to-body weight ratio in a dose-dependent manner and accelerates liver regeneration at early stage. C_LI Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/707802v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13b42feorg.highwire.dtl.DTLVardef@1ab6390org.highwire.dtl.DTLVardef@115c157org.highwire.dtl.DTLVardef@1486993_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epstein-Barr virus (EBV) latent infection is causally linked to several epithelial cancers, including endemic forms of undifferentiated nasopharyngeal carcinoma (NPC) and to a subtype of gastric cancer (GC). EBNA1 is the viral-encoded sequence-specific DNA-binding protein required for episome maintenance but also contributes to host-cell survival through multiple mechanisms including binding to host chromosome. We previously developed small molecule inhibitors of EBNA1 DNA-binding that block host cell cycle progression and growth of EBV+ tumors in vivo. However, the underlying molecular mechanisms of EBNA1 function and inhibition have not been completely elucidated. In this study, we employ VK1727 to inhibit EBNA1 DNA-binding to viral and cellular genomes in three EBV+ epithelial tumors (PDX C15, C666-1 and SNU719). We integrate EBNA1ChIP-seq and transcriptomic RNA-seq analyses to identify the cell cycle dependent kinase CDC7 and a stem cell transcription factor POU2F1 as direct functional targets of EBNA1 in these epithelial cancers. EBNA1 binding to CDC7 promoter and POU2F1 intron promotes RNA Pol II-pS5 to initiate transcription of these two genes. We show that CDC7 inhibitor Simurosertib is epistatic, while Bcl2 inhibitor Venetoclax is synergistic with VK1727 in the inhibition of EBV+ epithelial cancer cell proliferation and survival. Our study reveals new functional gene targets and pathways of VK1727 in EBV+ epithelial cancers that provide new biomarkers and combinatorial strategies to treat EBV-driven cancers. IMPORTANCEEBNA1 is essential for EBV latency and tumorigenesis, but its mechanism of action on host gene expression is not yet known. Small molecule inhibitors of EBNA1 DNA-binding block cell cycle progression and inhibit growth of EBV+ tumors. In this study, we use the EBNA1 small molecule inhibitor VK1727 to identify cellular gene targets that are bound by EBNA1 and deregulated by its pharmacological inhibition in EBV+ epithelial cancer cell lines and an NPC PDX mouse model. We identify cycle dependent kinase CDC7 and the stem cell transcription factor POU2F1 as EBNA1 bound and regulated genes important for EBV epithelial cancer proliferation. These findings not only decipher molecular mechanism how VK1727 blocks cell cycle progression and inhibits cell proliferation but also provide two new cellular gene targets and pathways for therapeutic intervention in EBV+ epithelial cancers.

A low-glucose microenvironment can induce metabolic abnormalities in tumour cells, including hepatocellular carcinoma (HCC) cells, and enhance cancer cell stemness. Isthmin-1 (ISM1) is a recently identified adipokine that promotes glucose uptake and enhances cellular metabolism. While the activity of the ISM1 protein is regulated by glycosylases, its transcriptional and posttranscriptional regulation remain poorly understood. A novel alternatively spliced variant of ISM1 (ISM1-AS) was recently identified. Unlike canonical ISM1, ISM1-AS lacks an AMOP domain, a key structural element required for ISM1 function, suggesting the loss of its metabolic regulatory activity. In this study, we found that ISM1 expression was significantly reduced in HCC tissues and correlated with poor prognosis. Functional assays revealed that ISM1 overexpression markedly suppressed HCC cell proliferation and invasion, whereas ISM1-AS overexpression had the opposite effect. Importantly, ISM1 co-overexpression attenuated the oncogenic effects of ISM1-AS. Knockdown of the antisense transcript lncRNA-ISM1 reduced ISM1-AS expression while increasing ISM1 expression, thereby suppressing HCC proliferation and migration. Mechanistically, lncRNA-ISM1 regulated ISM1 alternative splicing by interacting with RBM10, thereby altering the balance between ISM1-AS and ISM1. This shift activated the Akt-S6 signalling pathway, promoting glycolysis and HCC progression. In vivo experiments further confirmed that the lncRNA-ISM1/ISM1-AS/ISM1 axis drives tumour growth via Akt-S6 activation. Our findings demonstrate that lncRNA-ISM1 promotes HCC progression through the RBM10-mediated alternative splicing of ISM1 and activation of the Akt-S6 signalling pathway, highlighting its potential as a therapeutic target for HCC.

USP18 is a primary negative regulator of the type I interferon (IFN-I) signaling which regulates hundreds of IFN-stimulated genes for viral protection and anti-cancer immunity. USP18 plays dual roles in the IFN-I signaling: 1) deubiquitinase enzymatic function which cleaves ISG15 from its substrates and 2) scaffolding function through forming a complex with STAT2 to suppress IFN-I signaling. Targeting the scaffolding function of USP18, instead of its enzyme activity, is crucial for reducing cancer cell fitness and boosting anti-tumor immunity. However, the molecular basis of USP18s scaffolding function remains unclear due to the lack of structural information. Here, using a fusion tag strategy, we captured the transient USP18-STAT2 complex and determined a ternary complex structure of STAT2-USP18-ISG15 at 3.05 [A] resolution by cryogenic electron microscopy (cryo-EM) that delineated detailed USP18-STAT2 interactions. Remarkably, the ternary complex impairs USP18s enzymatic function by STAT2-mediated disruption of its catalytic triad. Structural analysis and mutagenesis identify specific USP18 point mutations, facilitating further investigation into the role of USP18 in IFN-I signaling. Taken together, our findings suggest that USP18s scaffolding function could present an untapped opportunity for cancer therapy.