Identification of lectin receptors for conserved SARS‐CoV‐2 glycosylation sites

New SARS‐CoV‐2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N‐glycan sites of Spike remain highly conserved among SARS‐CoV‐2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate‐binding proteins (lectins) to probe critical sugar residues on the full‐length trimeric Spike and the receptor binding domain (RBD) of SARS‐CoV‐2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single‐molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD‐ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS‐CoV‐2 infections. These data report the first extensive map and 3D structural modelling of lectin‐Spike interactions and uncovers candidate receptors involved in Spike binding and SARS‐CoV‐2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS‐CoV‐2 viral entry holds promise for pan‐variant therapeutic interventions.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

Molecular basis of cross‐species ACE2 interactions with SARS‐CoV‐2‐like viruses of pangolin origin

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS‐CoV‐2 causing the global COVID‐19 outbreak. Here, we study the binding of two SARS‐CoV‐2‐like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin‐converting enzyme 2 (hACE2), the receptor of SARS‐CoV‐2. We find that the spike protein receptor‐binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS‐CoV‐2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2‐expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS‐CoV‐2. Additionally, cryo‐EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS‐CoV‐2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS‐CoV‐2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.     

1‐Undecene from Pseudomonas aeruginosa is an olfactory signal for flight‐or‐fight response in Caenorhabditis elegans

Animals possess conserved mechanisms to detect pathogens and to improve survival in their presence by altering their own behavior and physiology. Here, we utilize Caenorhabditis elegans as a model host to ask whether bacterial volatiles constitute microbe‐associated molecular patterns. Using gas chromatography–mass spectrometry, we identify six prominent volatiles released by the bacterium Pseudomonas aeruginosa. We show that a specific volatile, 1‐undecene, activates nematode odor sensory neurons inducing both flight and fight responses in worms. Using behavioral assays, we show that worms are repelled by 1‐undecene and that this aversion response is driven by the detection of this volatile through AWB odor sensory neurons. Furthermore, we find that 1‐undecene odor can induce immune effectors specific to P. aeruginosa via AWB neurons and that brief pre‐exposure of worms to the odor enhances their survival upon subsequent bacterial infection. These results show that 1‐undecene derived from P. aeruginosa serves as a pathogen‐associated molecular pattern for the induction of protective responses in C. elegans.     

Aging‐related cell type‐specific pathophysiologic immune responses that exacerbate disease severity in aged COVID‐19 patients

Coronavirus disease 2019 (COVID‐19) is especially severe in aged patients, defined as 65 years or older, for reasons that are currently unknown. To investigate the underlying basis for this vulnerability, we performed multimodal data analyses on immunity, inflammation, and COVID‐19 incidence and severity as a function of age. Our analysis leveraged age‐specific COVID‐19 mortality and laboratory testing from a large COVID‐19 registry, along with epidemiological data of ~3.4 million individuals, large‐scale deep immune cell profiling data, and single‐cell RNA‐sequencing data from aged COVID‐19 patients across diverse populations. We found that decreased lymphocyte count and elevated inflammatory markers (C‐reactive protein, D‐dimer, and neutrophil–lymphocyte ratio) are significantly associated with age‐specific COVID‐19 severities. We identified the reduced abundance of naïve CD8 T cells with decreased expression of antiviral defense genes (i.e., IFITM3 and TRIM22) in aged severe COVID‐19 patients. Older individuals with severe COVID‐19 displayed type I and II interferon deficiencies, which is correlated with SARS‐CoV‐2 viral load. Elevated expression of SARS‐CoV‐2 entry factors and reduced expression of antiviral defense genes (LY6E and IFNAR1) in the secretory cells are associated with critical COVID‐19 in aged individuals. Mechanistically, we identified strong TGF‐beta‐mediated immune–epithelial cell interactions (i.e., secretory‐non‐resident macrophages) in aged individuals with critical COVID‐19. Taken together, our findings point to immuno‐inflammatory factors that could be targeted therapeutically to reduce morbidity and mortality in aged COVID‐19 patients.     

Disease‐linked TDP‐43 hyperphosphorylation suppresses TDP‐43 condensation and aggregation

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

4‐phenylpyridine suppresses UVB‐induced skin inflammation by targeting c‐Src in vitro and in vivo

Acute or repetitive exposure to ultraviolet (UV) cause disruptions to the skin barrier and subsequent inflammatory skin disease. 4‐phenylpyridine (4‐PP) is a constituent of Brassica campestris L. ssp. Pekinensis and its effect on skin inflammation and molecular target remain unclear. The purpose of this study is to confirm the anti‐inflammatory efficacy of 4‐PP on UVB‐induced skin inflammation in human keratinocytes HaCaT and mouse skin and validation of its molecular target. 4‐PP also attenuated UVB‐induced phosphorylation of p38/mitogen‐activated protein kinase kinase (MKK) 3/6, c‐Jun N‐terminal kinase 1/2, MKK 4/7, extracellular‐signal‐regulated kinase 1/2, mitogen‐activated protein kinase 1/2. Additionally, 4‐PP inhibited UVB‐induced phosphorylation of epidermal growth factor receptor (EGFR) Y1068, Y1045 and 854 residues but not the proto‐oncogene tyrosine‐protein kinase c‐Src. Drug affinity responsive target stability assay revealed that 4‐PP directly binds to c‐Src and inhibits pronase c‐proteolysis. Knockdown of c‐Src inhibited UVB‐induced COX‐2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells. Dorsal treatment of 4‐PP prevented UVB (0.5 J/cm²)‐induced skin thickness, phosphorylation of EGFR and COX‐2 expression in mouse skin. Our findings suggest that 4‐PP can be used as anti‐inflammatory agent with an effect of skin inflammation by inhibiting the COX‐2 expression via suppressing the c‐Src/EGFR/MAPKs signalling pathway.     

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

MicroRNA‐495‐3p diminishes doxorubicin‐induced cardiotoxicity through activating AKT

Doxorubicin (Dox) is a broad‐spectrum antitumour agent; however, its clinical application is impeded due to the cumulative cardiotoxicity. The present study aims to investigate the role and underlying mechanisms of microRNA‐495‐3p (miR‐495‐3p) in Dox‐induced cardiotoxicity. Herein, we found that cardiac miR‐495‐3p expression was significantly decreased in Dox‐treated hearts, and that the miR‐495‐3p agomir could prevent oxidative stress, cell apoptosis, cardiac mass loss, fibrosis and cardiac dysfunction upon Dox stimulation. In contrast, the miR‐495‐3p antagomir dramatically aggravated Dox‐induced cardiotoxicity in mice. Besides, we found that the miR‐495‐3p agomir attenuated, while the miR‐495‐3p antagomir exacerbated Dox‐induced oxidative stress and cellular injury in vitro. Mechanistically, we demonstrated that miR‐495‐3p directly bound to the 3′‐untranslational region of phosphate and tension homology deleted on chromosome ten (PTEN), downregulated PTEN expression and subsequently activated protein kinase B (PKB/AKT) pathway, and that PTEN overexpression or AKT inhibition completely abolished the cardioprotective effects of the miR‐495‐3p agomir. Our study for the first time identify miR‐495‐3p as an endogenous protectant against Dox‐induced cardiotoxicity through activating AKT pathway in vivo and in vitro.     

Effector‐dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi‐amr3 and Rpi‐amr1

Plant pathogens compromise crop yields. Plants have evolved robust innate immunity that depends in part on intracellular Nucleotide‐binding, Leucine rich‐Repeat (NLR) immune receptors that activate defense responses upon detection of pathogen‐derived effectors. Most “sensor” NLRs that detect effectors require the activity of “helper” NLRs, but how helper NLRs support sensor NLR function is poorly understood. Many Solanaceae NLRs require NRC (NLR‐Required for Cell death) class of helper NLRs. We show here that Rpi‐amr3, a sensor NLR from Solanum americanum, detects AVRamr3 from the potato late blight pathogen, Phytophthora infestans, and activates oligomerization of helper NLRs NRC2 and NRC4 into high‐molecular‐weight resistosomes. In contrast, recognition of P. infestans effector AVRamr1 by another sensor NLR Rpi‐amr1 induces formation of only the NRC2 resistosome. The activated NRC2 oligomer becomes enriched in membrane fractions. ATP‐binding motifs of both Rpi‐amr3 and NRC2 are required for NRC2 resistosome formation, but not for the interaction of Rpi‐amr3 with its cognate effector. NRC2 resistosome can be activated by Rpi‐amr3 upon detection of AVRamr3 homologs from other Phytophthora species. Mechanistic understanding of NRC resistosome formation will underpin engineering crops with durable disease resistance.     

Cyclic di‐GMP modulates sessile‐motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice‐growing countries. We showed recently that the universal bacterial second messenger c‐di‐GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c‐di‐GMP receptor domain, known as the PilZ‐domain. By deleting all the genes encoding c‐di‐GMP‐degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c‐di‐GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild‐type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c‐di‐GMP, which together play a critical role in regulating the c‐di‐GMP‐associated functionality. The findings from this study fill a gap in our understanding of how c‐di‐GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.     

Nonproductive exposure of PBMCs to SARS‐CoV‐2 induces cell‐intrinsic innate immune responses

Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.     

Insulin binding to the analytical antibody sandwich pair OXI‐005 and HUI‐018: Epitope mapping and binding properties

The insulin epitopes for two monoclonal antibodies (mAbs), OXI‐005 and HUI‐018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X‐ray crystallography. The crystal structure of the HUI‐018 Fab in complex with human insulin (HI) was determined and OXI‐005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI‐005 epitope comprises insulin residues 1,3,4,19–21 (A‐chain) and 25–30 (B‐chain) and for HUI‐018 residues 7,8,10–14,17 (A‐chain) and 5–7, 10, 14 (B‐chain). The areas of insulin involved in interactions with the mAb are 20% (OXI‐005) and 24% (HUI‐018) of the total insulin surface. Based on the Fab complex crystal structures with the insulins a molecular model for simultaneous binding of the Fabs to PI was built and this model was validated by small angle X‐ray scattering measurements for the ternary complex. The epitopes for the mAbs on insulin were found well separated from each other as expected from luminiscent oxygen channeling immunoassay results for different insulins (HI, PI, bovine insulin, DesB30 HI, insulin glargine, insulin lispro). The affinities of the OXI‐005 and HUI‐018 Fabs for HI, PI, and DesB30 HI were determined using surface plasmon resonance. The K _Ds were found to be in the range of 1–4 nM for the HUI‐018 Fab, while more different for the OXI‐005 Fab (50 nM for HI, 20 nM for PI and 400 nM for DesB30 HI) supporting the importance of residue B30 for binding to OXI‐005.     

Structural investigation of CDCA3‐Cdh1 protein–protein interactions using in vitro studies and molecular dynamics simulation

The anaphase‐promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell‐cycle proteins and their functions during mitosis. Levels of the protein cell division cycle‐associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/C^Cdh1. CDCA3 is an intrinsically disordered protein and contains both C‐terminal KEN box and D‐box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation‐dependent non‐canonical ABBA‐like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA‐like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H‐bonds, hydrophobic and ionic interactions across the CDCA3 ABBA‐like motif in the linker region between KEN and D‐box motifs. This linker region adopts a well‐defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non‐canonical ABBA‐like motif to promote binding to the APC/C^Cdh1 and regulation of CDCA3 protein levels.     

Solution structures of the Shewanella woodyi H‐NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP‐051

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.     

METTL3‐mediated maturation of miR‐589‐5p promotes the malignant development of liver cancer

MiR‐589‐5p could promote liver cancer, but the specific mechanisms are largely unknown. This study examined the role and mechanisms of miR‐589‐5p in liver cancer. The expressions of miR‐589‐5p, METTL3 and m6A in liver cancers were determined by RT‐qPCR. The relationship between miR‐589‐5p and METTL3‐mediated m6A methylation was examined by m6A RNA immunoprecipitation. After transfection, the viability, migration, invasion and expressions of METTL3 and miR‐589‐5p in liver cancer cells were detected by CCK‐8, wound‐healing, transwell and RT‐qPCR. After the xenograft tumour was established in mice, the tumour volume was determined and the expressions of METTL3, miR‐589‐5p, MMP‐2, TIMP‐2, E‐cadherin, N‐cadherin and Vimentin in tumour tissue were detected by RT‐qPCR and Western blotting. In vitro study showed that miR‐589‐5p and METTL3 were highly expressed in liver cancer. METTL3 was positively correlated with miR‐589‐5p. METTL3 up‐regulated the expression of miR‐589‐5p and promoted the maturation of miR‐589‐5p. Overexpressed miR‐589‐5p and METTL3 promoted the viability, migration and invasion of liver cancer cells, while the effects of silencing miR‐589‐5p and METTL3 on the cells were the opposite. The effects of METTL3 overexpression and silencing were reversed by miR‐589‐5p inhibitor and mimic, respectively. In vivo study showed that METLL3 silencing inhibited the growth of xenograft tumour and the expressions of METTL3, MMP‐2, N‐cadherin and Vimentin, promoted the expressions of TIMP‐2 and E‐cadherin, while miR‐589‐5p mimic caused the opposite results and further reversed the effects of METLL3 silencing. In summary, this study found that METTL3‐mediated maturation of miR‐589‐5p promoted the malignant development of liver cancer.