rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly.

Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain (spb1D52A) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF4- transition state structure that implicates the direct involvement of unmodified G2922 in Nog2 GTPase activation. Genetic suppressors and in vivo imaging indicate that premature GTP hydrolysis prevents the efficient binding of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 recruitment to the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Our approach and findings provide a template to study the GTPase cycles and regulatory factor interactions of the other K-loop GTPases involved in ribosome assembly.

The model diatom Phaeodactylum tricornutum provides insights into the diversity and function of microeukaryotic DNA methyltransferases.

Cytosine methylation is an important epigenetic mark involved in the transcriptional control of transposable elements in mammals, plants and fungi. The Stramenopiles-Alveolate-Rhizaria (SAR) lineages are a major group of ecologically important marine microeukaryotes, including the phytoplankton groups diatoms and dinoflagellates. However, little is known about their DNA methyltransferase diversity. Here, we performed an in-silico analysis of DNA methyltransferases found in marine microeukaryotes and showed that they encode divergent DNMT3, DNMT4, DNMT5 and DNMT6 enzymes. Furthermore, we found three classes of enzymes within the DNMT5 family. Using a CRISPR/Cas9 strategy we demonstrated that the loss of the DNMT5a gene correlates with a global depletion of DNA methylation and overexpression of young transposable elements in the model diatom Phaeodactylum tricornutum. The study provides a view of the structure and function of a DNMT family in the SAR supergroup using an attractive model species.

Structure, activity and function of the lysine methyltransferase SETD5.

SET domain-containing 5 (SETD5) is an uncharacterized member of the protein lysine methyltransferase family and is best known for its transcription machinery by methylating histone H3 on lysine 36 (H3K36). These well-characterized functions of SETD5 are transcription regulation, euchromatin formation, and RNA elongation and splicing. SETD5 is frequently mutated and hyperactive in both human neurodevelopmental disorders and cancer, and could be down-regulated by degradation through the ubiquitin-proteasome pathway, but the biochemical mechanisms underlying such dysregulation are rarely understood. Herein, we provide an update on the particularities of SETD5 enzymatic activity and substrate specificity concerning its biological importance, as well as its molecular and cellular impact on normal physiology and disease, with potential therapeutic options.

Loss of the m6A methyltransferase METTL3 in monocyte-derived macrophages ameliorates Alzheimer's disease pathology in mice.

Alzheimer's disease (AD) is a heterogeneous disease with complex clinicopathological characteristics. To date, the role of m6A RNA methylation in monocyte-derived macrophages involved in the progression of AD is unknown. In our study, we found that methyltransferase-like 3 (METTL3) deficiency in monocyte-derived macrophages improved cognitive function in an amyloid beta (Aß)-induced AD mouse model. The mechanistic study showed that that METTL3 ablation attenuated the m6A modification in DNA methyltransferase 3A (Dnmt3a) mRNAs and consequently impaired YTH N6-methyladenosine RNA binding protein 1 (YTHDF1)-mediated translation of DNMT3A. We identified that DNMT3A bound to the promoter region of alpha-tubulin acetyltransferase 1 (Atat1) and maintained its expression. METTL3 depletion resulted in the down-regulation of ATAT1, reduced acetylation of α-tubulin and subsequently enhanced migration of monocyte-derived macrophages and Aß clearance, which led to the alleviated symptoms of AD. Collectively, our findings demonstrate that m6A methylation could be a promising target for the treatment of AD in the future.

Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition.

Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells.

Knockout of DDM1 in Physcomitrium patens disrupts DNA methylation with a minute effect on transposon regulation and development.

The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a non-flowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.

Caprin-1 plays a role in cell proliferation and Warburg metabolism of esophageal carcinoma by regulating METTL3 and WTAP.

BACKGROUND: Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is implicated in cancer cell proliferation and tumorigenesis; however, its role in the development of esophageal carcinoma (ESCA) has not been examined. METHODS: Biological methods and data analysis were used to investigate the expression of Caprin-1 in ESCA tissue and cell lines. We comprehensively analyzed the mRNA expression and prognostic values, signalling pathways of CAPRIN1 in ESCA using public databases online. Biological functions of CAPRIN1 were performed by clorimetric growth assay, EdU staining, colony formation, flow cytometry, apoptosis analysis, Western blot, lactate detection assay, extracellular acidification rates. The underlying mechanism was determined via flow cytometric analysis, Western blot and rescue experiments. In addition, xenograft tumor model was constructed to verify the phenotypes upon CAPRIN1 silencing. RESULTS: Caprin-1 expression was significantly elevated in both ESCA tumor tissues and cell lines compared with that in normal adjacent tissues and fibroblasts. Increased CAPRIN1 mRNA expression was significantly associated with clinical prognosis and diagnostic accuracy. The GO enrichment and KEGG pathway analysis CAPRIN1 might be related to immune-related terms, protein binding processes, and metabolic pathways. A significant positive correlation was observed between high Caprin-1 protein levels and lymph node metastasis (P = 0.031), ki-67 (P = 0.023), and 18F- FDG PET/CT parameters (SUVmax (P = 0.002) and SUV mean (P = 0.005)) in 55 ESCA patients. At cut-off values of SUVmax 17.71 and SUVmean 10.14, 18F- FDG PET/CT imaging predicted Caprin-1 expression in ESCA samples with 70.8% sensitivity and 77.4% specificity. In vitro and in vivo assays showed that Caprin-1 knockdown affected ESCA tumor growth. Silencing Caprin-1 inhibited ESCA cell proliferation and glycolysis, and decreased the expression of methyltransferase-like 3 (METTL3) and Wilms' tumor 1-associating protein (WTAP). However, this effect could be partially reversed by the restoration of METTL3 and WTAP expression. CONCLUSIONS: Our data suggest that Caprin-1 could serve as a prognostic biomarker and has an oncogenic role in ESCA.

The RNA m5C Methylase NSUN2 Modulates Corneal Epithelial Wound Healing.

Purpose: The emerging epitranscriptomics offers insights into the physiopathological roles of various RNA modifications. The RNA methylase NOP2/Sun domain family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modification of mRNAs. However, the role of NSUN2 in corneal epithelial wound healing (CEWH) remains unknown. Here we describe the functional mechanisms of NSUN2 in mediating CEWH. Methods: RT-qPCR, Western blot, dot blot, and ELISA were used to determine the NSUN2 expression and overall RNA m5C level during CEWH. NSUN2 silencing or overexpression was performed to explore its involvement in CEWH both in vivo and in vitro. Multi-omics was integrated to reveal the downstream target of NSUN2. MeRIP-qPCR, RIP-qPCR, and luciferase assay, as well as in vivo and in vitro functional assays, clarified the molecular mechanism of NSUN2 in CEWH. Results: The NSUN2 expression and RNA m5C level increased significantly during CEWH. NSUN2 knockdown significantly delayed CEWH in vivo and inhibited human corneal epithelial cells (HCEC) proliferation and migration in vitro, whereas NSUN2 overexpression prominently enhanced HCEC proliferation and migration. Mechanistically, we found that NSUN2 increased ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) translation through the binding of RNA m5C reader Aly/REF export factor. Accordingly, UHRF1 knockdown significantly delayed CEWH in vivo and inhibited HCEC proliferation and migration in vitro. Furthermore, UHRF1 overexpression effectively rescued the inhibitory effect of NSUN2 silencing on HCEC proliferation and migration. Conclusions: NSUN2-mediated m5C modification of UHRF1 mRNA modulates CEWH. This finding highlights the critical importance of this novel epitranscriptomic mechanism in control of CEWH.

Fate of arsenicals in mice carrying the human AS3MT gene exposed to environmentally relevant levels of arsenite in drinking water.

Although mice are widely used to study adverse effects of inorganic arsenic (iAs), higher rates of iAs methylation in mice than in humans may limit their utility as a model organism. A recently created 129S6 mouse strain in which the Borcs7/As3mt locus replaces the human BORCS7/AS3MT locus exhibits a human-like pattern of iAs metabolism. Here, we evaluate dosage dependency of iAs metabolism in humanized (Hs) mice. We determined tissue and urinary concentrations and proportions of iAs, methylarsenic (MAs), and dimethylarsenic (DMAs) in male and female Hs and wild-type (WT) mice that received 25- or 400-ppb iAs in drinking water. At both exposure levels, Hs mice excrete less total arsenic (tAs) in urine and retain more tAs in tissues than WT mice. Tissue tAs levels are higher in Hs females than in Hs males, particularly after exposure to 400-ppb iAs. Tissue and urinary fractions of tAs present as iAs and MAs are significantly greater in Hs mice than in WT mice. Notably, tissue tAs dosimetry in Hs mice resembles human tissue dosimetry predicted by a physiologically based pharmacokinetic model. These data provide additional support for use of Hs mice in laboratory studies examining effects of iAs exposure in target tissues or cells.

Prospects and feasibility of synergistic therapy with radiotherapy, immunotherapy, and DNA methyltransferase inhibitors in non-small cell lung cancer.

The morbidity and mortality of lung cancer are increasing, seriously threatening human health and life. Non-small cell lung cancer (NSCLC) has an insidious onset and is not easy to be diagnosed in its early stage. Distant metastasis often occurs and the prognosis is poor. Radiotherapy (RT) combined with immunotherapy, especially with immune checkpoint inhibitors (ICIs), has become the focus of research in NSCLC. The efficacy of immunoradiotherapy (iRT) is promising, but further optimization is necessary. DNA methylation has been involved in immune escape and radioresistance, and becomes a game changer in iRT. In this review, we focused on the regulation of DNA methylation on ICIs treatment resistance and radioresistance in NSCLC and elucidated the potential synergistic effects of DNA methyltransferases inhibitors (DNMTis) with iRT. Taken together, we outlined evidence suggesting that a combination of DNMTis, RT, and immunotherapy could be a promising treatment strategy to improve NSCLC outcomes.

OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli.

O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.

The construction of a novel ferroptosis-related lncRNA model to predict prognosis in colorectal cancer patients.

Colorectal cancer (CRC) is the most common gastrointestinal tumor with poor prognosis. Ferroptosis is a pivotal form of programmed iron-dependent cell death different from autophagy and apoptosis, and long noncoding RNA (lncRNA) can influence the prognosis of CRC via regulating ferroptosis. To explore the role and prognostic value of the constructed ferroptosis-related lncRNA model in CRC, a prognostic model was constructed and validated by screening ferroptosis-related lncRNAs associated with prognosis based on the transcriptome data and survival data of CRC patients in The Cancer Genome Atlas database. Regarding the established prognostic models, differences in signaling pathways and immune infiltration, as well as differences in immune function, immune checkpoints, and N6-methyladenosine-related genes were also analyzed. A total of 6 prognostic ferroptosis-related lncRNAs were obtained, including AP003555.1, AC010973.2, LINC01857, AP001469.3, ITGB1-DT and AC129492.1. Univariate independent prognostic analysis, multivariate independent prognostic analysis and receiver operating characteristic curves showed that ferroptosis-related lncRNAs could be recognized as independent prognostic factors. The Kaplan-Meier survival curves and the risk curves showed that the survival time of the high-risk group was shorter. Gene set enrichment analysis enrichment analysis showed that ATP-binding cassette transporters, taste transduction and VEGF signaling pathway were more active in high-risk groups that than in low-risk groups. However, the citrate cycle tricarboxylic acid cycle, fatty acid metabolism and peroxisome were significantly more active in the low-risk group than in the high-risk group. In addition, there were also differences in immune infiltration in the high-low-risk groups based on different methods, including antigen-presenting cell co-stimulation, chemokine receptor, parainflammation, and Type II IFN Response. Further analysis of Immune checkpoints showed that most of the Immune checkpoints such as TNFRSF18, LGALS9 and CTLA4 in the high-risk group were significantly higher than those in the low-risk group, and the expressions of N6-methyladenosine related genes METTL3, YTHDH2 and YTHDC1 were also significantly different in the high-risk group. Ferroptosis-related lncRNAs are closely related to the survival of colorectal cancer patients, which can be used as new biomarkers and potential therapeutic targets for the prognosis of colorectal cancer.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

METTL3 enhances the effect of YTHDF1 on NEDD1 mRNA stability by m6A modification in diffuse large B-cell lymphoma cells.

AIM: Diffuse large B-cell lymphoma (DLBCL) remains the most frequent subpopulation of lymphoma, and N6-methyladenosine (m6A) was implicated in the DLBCL progression. Herein, we sought to decipher the m6A-asociated mechanism of NEDD1 in DLBCL development. METHODS: The NEDD1 expression profile in DLBCL was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot. NEDD1 was artificially downregulated or upregulated in DLBCL cells, followed by EdU, Transwell assays and flow cytometry. The Hedgehog pathway activity was assayed by a dual-luciferase assay. The m6A methylation of NEDD1 in DLBCL was assessed by meRIP-qPCR, and the regulatory mechanism of METTL3 on NEDD1 was validated. The LDH assay was conducted to examine the impact of CD8+ T cells on DLBCL cells. The DLBCL cells were administrated into mice to evaluate the tumorigenic activity and ki-67 activity in tumor tissues. RESULTS: NEDD1 was overexpressed in DLBCL. Depletion of NEDD1 inhibited the aggressiveness of SU-DHL-8 and OCI-LY1 cells, whereas overexpression of NEDD1 expedited the aggressiveness of SU-DHL-8 and OCI-LY1 cells. METTL3 promoted NEDD1 translation in an m6A-dependent manner via YTHDF1. Depletion of METTL3 inhibited SU-DHL-8 and OCI-LY1 cell activity through regulation of NEDD1. NEDD1 reversed the repressive effect of METTL3 loss on the aggressiveness of SU-DHL-8 and OCI-LY1 cells. NEDD1 activated the Hedgehog signaling to promote immune escape of DLBCL. CONCLUSIONS: METTL3 promotes translation of NEDD1 via YTHDF1-depedndent m6A modification, thereby activating the Hedgehog signaling pathway to promote immune escape of DLBCL cells.

CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori.

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.

Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

BACKGROUND: With the increasing morbidity and mortality of preeclampsia (PE), it has posed a huge challenge to public health. Previous studies have reported endoplasmic reticulum (ER) stress could contribute to trophoblastic dysfunction which was associated with the N6-methyladenosine (m6A) modification by methyltransferase-like 3 (METTL3), resulting in PE. However, little was known about the relationship between METTL3 and ER stress in PE. Thus, in vitro and in vivo studies were performed to clarify the mechanism about how METTL3 affects the trophoblasts under ER stress in PE and to explore a therapeutic approach for PE. METHODS: An ER stress model in HTR-8/SVneo cells and a preeclamptic rat model were used to study the mechanism and explore a therapeutic approach for PE. Western blot, immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and methylated RNA immunoprecipitation (MeRIP)-qPCR were performed to detect the protein, RNA, and methylated transmembrane BAX inhibitor motif containing 6 (TMBIM6) expression levels. The m6A colorimetric and mRNA stability assays were used to measure the m6A levels and TMBIM6 stability, respectively. Short hairpin RNAs (shRNAs) were used to knockdown METTL3 and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Flow cytometry and Transwell assays were performed to evaluate the apoptosis and invasion abilities of trophoblasts. RESULTS: Upregulated METTL3 and m6A levels and downregulated TMBIM6 levels were observed in preeclamptic placentas under ER stress. The ER stress model was successfully constructed, and knockdown of METTL3 had a beneficial effect on HTR-8/SVneo cells under ER stress as it decreased the levels of methylated TMBIM6 mRNA. Moreover, overexpression of TMBIM6 was beneficial to HTR-8/SVneo cells under ER stress as it could neutralize the harmful effects of METTL3 overexpression. Similar to the knockdown of METTL3, downregulation of YTHDF2 expression resulted in the increased expression and mRNA stability of TMBIM6. Finally, improved systemic symptoms as well as protected placentas and fetuses were demonstrated in vivo. CONCLUSIONS: METTL3/YTHDF2/TMBIM6 axis exerts a significant role in trophoblast dysfunction resulting in PE while inhibiting METTL3 may provide a novel therapeutic approach for PE.

METTL3 depletion contributes to tumour progression and drug resistance via N6 methyladenosine-dependent mechanism in HR+HER2-breast cancer.

BACKGROUND: Chemotherapy is an important strategy for the treatment of hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+HER2-) breast cancer (BC), but this subtype has a low response rate to chemotherapy. Growing evidence indicates that N6-methyladenosine (m6A) is the most common RNA modification in eukaryotic cells and that methyltransferase-like 3 (METTL3) participates in tumour progression in several cancer types. Therefore, exploring the function of METTL3 in HR+HER2- BC initiation and development is still important. METHODS: mRNA and protein expression levels were analysed by quantitative real-time polymerase chain reaction and western blotting, respectively. Cell proliferation was detected by CCK-8 and colony formation assays. Cell cycle progression was assessed by flow cytometry. Cell migration and invasion were analysed by wound healing assays and transwell assays, respectively, and apoptosis was analysed by TUNEL assays. Finally, m6A modification was analysed by methylated RNA immunoprecipitation. RESULTS: Chemotherapy-induced downregulation of the m6A modification is regulated by METTL3 depletion in HR+HER2- BC. METTL3 knockdown in MCF-7/T47D cells decreased the drug sensitivity of HR+HER2- BC cells by promoting tumour proliferation and migration and inhibiting apoptosis. Mechanistically, CDKN1A is a downstream target of METTL3 that activates the AKT pathway and promotes epithelial-mesenchymal transformation (EMT). Moreover, a decrease in BAX expression was observed when m6A modification was inhibited with METTL3 knockdown, and apoptosis was inhibited by the reduction of caspase-3/-9/-8. CONCLUSION: METTL3 depletion promotes the proliferation and migration and decreases the drug sensitivity of HR+HER2- BC via regulation of the CDKN1A/EMT and m6A-BAX/caspase-9/-3/-8 signalling pathways, which suggests METTL3 played a tumour-suppressor role and it could be a potential biomarker for predicting the prognosis of patients with HR+HER2- BC.

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

N6-methyladenosine modification in 18S rRNA promotes tumorigenesis and chemoresistance via HSF4b/HSP90B1/mutant p53 axis.

Aberrant N6-methyladenosine (m6A) modification on mRNA is correlated with cancer progression. However, the role of m6A on ribosomal RNA (rRNA) in cancer remains poorly understood. Our current study reveals that METTL5/TRMT112 and their mediated m6A modification at the 18S rRNA 1832 site (m6A1832) are elevated in nasopharyngeal carcinoma (NPC) and promote oncogenic transformation in vitro and in vivo. Moreover, loss of catalytic activity of METTL5 abolishes its oncogenic functions. Mechanistically, m6A1832 18S rRNA modification facilitates the assembly of 80S ribosome via bridging the RPL24-18S rRNA interaction, therefore promoting the translation of mRNAs with 5' terminal oligopyrimidine (5' TOP) motifs. Further mechanistic analysis reveals that METTL5 enhances HSF4b translation to activate the transcription of HSP90B1, which binds with oncogenic mutant p53 (mutp53) protein and prevents it from undergoing ubiquitination-dependent degradation, therefore facilitating NPC tumorigenesis and chemoresistance. Overall, our findings uncover an innovative mechanism underlying rRNA epigenetic modification in regulating mRNA translation and the mutp53 pathway in cancer.

m5C-dependent cross-regulation between nuclear reader ALYREF and writer NSUN2 promotes urothelial bladder cancer malignancy through facilitating RABL6/TK1 mRNAs splicing and stabilization.

The significance of 5-methylcytosine (m5C) methylation in human malignancies has become an increasing focus of investigation. Here, we show that m5C regulators including writers, readers and erasers, are predominantly upregulated in urothelial carcinoma of the bladder (UCB) derived from Sun Yat-sen University Cancer Center and The Cancer Genome Atlas cohort. In addition, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a methyltransferase and Aly/REF export factor (ALYREF) as a nuclear m5C reader, are frequently coexpressed in UCB. By applying patient-derived organoids model and orthotopic xenograft mice model, we demonstrate that ALYREF enhances proliferation and invasion of UCB cells in an m5C-dependent manner. Integration of tanscriptome-wide RNA bisulphite sequencing (BisSeq), RNA-sequencing (RNA-seq) and RNA Immunoprecipitation (RIP)-seq analysis revealed that ALYREF specifically binds to hypermethylated m5C site in RAB, member RAS oncogene family like 6 (RABL6) and thymidine kinase 1 (TK1) mRNA via its K171 domain. ALYREF controls UCB malignancies through promoting hypermethylated RABL6 and TK1 mRNA for splicing and stabilization. Moreover, ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB. Clinically, the patients with high coexpression of ALYREF/RABL6/TK1 axis had the poorest overall survival. Our study unveils an m5C dependent cross-regulation between nuclear reader ALYREF and m5C writer NSUN2 in activation of hypermethylated m5C oncogenic RNA through promoting splicing and maintaining stabilization, consequently leading to tumor progression, which provides profound insights into therapeutic strategy for UCB.