Protein function alteration and protein mislocalization are cancer hallmarks that drive oncogenesis. N6-methyladenosine (m6A) deposition mediated by METTL3, METTL16, and METTL5 together with the contribution of additional subunits of the m6A system, has shown a dramatic impact on cancer development. However, the cellular localization of m6A proteins inside tumor cells has been little studied so far. Interestingly, recent evidence indicates that m6A methyltransferases are not always confined to the nucleus, suggesting that epitranscriptomic factors may also have multiple oncogenic roles beyond m6A that still represent an unexplored field. To date novel epigenetic drugs targeting m6A modifiers, such as METTL3 inhibitors, are entering into clinical trials, therefore, the study of the potential onco-properties of m6A effectors beyond m6A is required. Here we will provide an overview of methylation-independent functions of the m6A players in cancer, describing the molecular mechanisms involved and the future implications for therapeutics.

Methyltransferases are a wide-ranging, yet well-conserved, class of molecules that have been found to modify a wide variety of substrates. Interest in RNA methylation has surged in recent years with the identification of the major eukaryotic mRNA m6A methyltransferase METTL3. METTL16 has also been identified as an RNA m6A methyltransferase; however, much less is known about its targets and actions. Interestingly, in addition to their catalytic activities, both METTL3 and METTL16 also have \"methylation-independent\" functions, including translational regulation, which have been discovered. However, evidence suggests that METTL16\'s role as an RNA-binding protein may be more significant than is currently recognized. In this review, we will introduce RNA methylation, specifically m6A, and the enzymes responsible for its deposition. We will discuss the varying roles that these enzymes perform and delve deeper into their RNA targets and possible roles as methylation-independent RNA binding proteins. Finally, we will touch upon the many open questions still remaining.

N6-methyladenosine (m6A) is one of the most prevalent and conserved RNA modifications. It controls several biological processes, including the biogenesis and function of circular RNAs (circRNAs), which are a class of covalently closed-single stranded RNAs. Several studies have revealed that proteotoxic stress response induction could be a relevant anticancer therapy in Acute Myeloid Leukemia (AML). Furthermore, a strong molecular interaction between the m6A mRNA modification factors and the suppression of the proteotoxic stress response has emerged. Since the proteasome inhibition leading to the imbalance in protein homeostasis is strictly linked to the stress response induction, we investigated the role of Bortezomib (Btz) on m6A regulation and in particular its impact on the modulation of m6A-modified circRNAs expression. Here, we show that treating AML cells with Btz downregulated the expression of the m6A regulator WTAP at translational level, mainly because of increased oxidative stress. Indeed, Btz treatment promoted oxidative stress, with ROS generation and HMOX-1 activation and administration of the reducing agent N-acetylcysteine restored WTAP expression. Additionally, we identified m6A-modified circRNAs modulated by Btz treatment, including circHIPK3, which is implicated in protein folding and oxidative stress regulation. These results highlight the intricate molecular networks involved in oxidative and ER stress induction in AML cells following proteotoxic stress response, laying the groundwork for future therapeutic strategies targeting these pathways.

BACKGROUND: KIAA1429, a regulatory subunit of the N6-methyladenosine (m6A) methyltransferase complex, has been implicated in the progression of various cancers. However, the role of KIAA1429 in gastric cancer (GC) and its underlying mechanisms remain elusive. This study aimed to investigate the role of KIAA1429 in GC and to elucidate the underlying mechanisms.
METHODS: The expression patterns and clinical relevance of KIAA1429 in GC were assessed using quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and bioinformatic analysis. In vitro and in vivo loss- and gain-of-function assays, m6A dot blot assays, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, dual luciferase reporter assays, RNA stability assays, RNA immunoprecipitation (RIP) assays, and RNA pull-down assays were performed to investigate the biological functions and underlying molecular mechanisms of KIAA1429 in GC.
RESULTS: Both the mRNA and protein expression of KIAA1429 were greater in GC tissues than in normal gastric tissues. High KIAA1429 expression correlated positively with poor prognosis in GC patients. KIAA1429 not only promoted GC cell proliferation, colony formation, G2/M cell cycle transition, migration, and invasion in vitro but also enhanced GC tumor growth and metastasis in vivo. Mechanistically, KIAA1429 increased the m6A level of RASD1 mRNA and enhanced its stability in an m6A-YTHDF2-dependent manner, thereby upregulating its expression. RASD1 knockdown partially rescued the KIAA1429 knockdown-induced impairment of pro‑oncogenic ability in GC cells. The expression levels of KIAA1429 and RASD1 were negatively correlated in GC tissues.
CONCLUSIONS: KIAA1429 plays a pro‑oncogenic role in GC by downregulating RASD1 expression through destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner. KIAA1429 may serve as a prognostic biomarker and therapeutic target for GC.

m6A methylation, a ubiquitous modification on circRNAs, exerts a profound influence on RNA function, intracellular behavior, and diverse biological processes, including disease development. While prediction algorithms exist for mRNA m6A modifications, a critical gap remains in the prediction of circRNA m6A modifications. Therefore, accurate identification and prediction of m6A sites are imperative for understanding RNA function and regulation. This study presents a novel hybrid model combining a convolutional neural network (CNN) and a bidirectional long short-term memory network (BiLSTM) for precise m6A methylation site prediction in circular RNAs (circRNAs) based on data from HEK293 cells. This model exploits the synergy between CNN\'s ability to extract intricate sequence features and BiLSTM\'s strength in capturing long-range dependencies. Furthermore, the integrated attention mechanism empowers the model to pinpoint critical biological information for studying circRNA m6A methylation. Our model, exhibiting over 78% prediction accuracy on independent datasets, offers not only a valuable tool for scientific research but also a strong foundation for future biomedical applications. This work not only furthers our understanding of gene expression regulation but also opens new avenues for the exploration of circRNA methylation in biological research.

OBJECTIVE: Breast cancer (BC) is a cancer that seriously affects women\'s health. BC cell migration increases the mortality of BC patients. Current studies have shown that long noncoding RNAs (LncRNAs) are related to the metastasis mechanism of BC. This study aimed to explore the function and role of LncRNA OIP5-AS1 in BC. And we analyzed its regulatory mechanism and related modification process.
METHODS: Our study analyzed the expression pattern of OIP5-AS1 in BC tissues and cell lines by qRT-PCR. The effects of OIP5-AS1 on the function of BC cells were detected by CCK-8 and transwell experiments. Bioinformatics analysis and double luciferase reporter gene detection were used to confirm the correlation between OIP5-AS1 and miR-150-5p and between miR-150-5p and Cyclin D2 (CCND2). The rescue test analyzed the effect of miR-150-5p regulating OIP5-AS1. In addition, the N6-methyladenosine (m6A) modification process of OIP5-AS1 was analyzed by RNA m6A dot blot, RIP assay, and double luciferase report experiment.
RESULTS: OIP5-AS1 was significantly upregulated in BC tissues and cell lines. OIP5-AS1 knockdown inhibited BC cell viability, migration and invasion. OIP5-AS1 upregulated CCND2 by binding with miR-150-5p. This process affected the metastasis of BC. Higher degree of m6A methylation was confirmed in BC cell lines. There were some binding sites between methyltransferase like 3 (METTL3) and OIP5-AS1. Moreover, the silencing of METTL3 inhibited the OIP5-AS1 expression through decreasing the m6A methylation levels.
CONCLUSIONS: LncRNA OIP5-AS1 promoted cell viability and metastasis of BC cells by targeting miR-150-5p/CCND2 axis. This process was modified by m6A methylation of METTL3.

Pancreatic cancer (PC) responds weakly to conventional immunotherapy. RNA N6-methyladenosine (m6A) modification has an essential role in the immune response, while its potential role in PC tumor microenvironment (TME) immune cell infiltration remains unknown. In this study, we thoroughly assessed the m6A modification patterns of 472 PC samples using 19 m6A regulators, and we systematically correlated these modification patterns with TME immune cell infiltration characteristics. We also created the m6Ascore and evaluated the m6A modification patterns of individual tumors, identified three different m6A modification patterns, and explored the role of the important m6A \"writer\" RBM15 in the regulation of macrophage function in PC. Two independent PC cohorts confirmed that patients with higher m6Ascore showed significant survival benefit. We verified that knockdown of RBM15 has the ability to inhibit PC growth and to promote macrophage infiltration and enhance phagocytosis of PC cells by macrophages. In conclusion, m6A modifications play a non-negligible role in the formation of TME diversity and complexity in PC. We reveal that inhibition of RBM15 suppresses PC development and modulates macrophage phagocytosis, and provide a more effective immunotherapeutic strategy for PC.

Epitranscriptomics represents a further layer of gene expression regulation. Specifically, N6-methyladenosine (m6A) regulates RNA maturation, stability, degradation, and translation. Regarding microRNAs (miRNAs), while it has been reported that m6A impacts their biogenesis, the functional effects on mature miRNAs remain unclear. Here, we show that m6A modification on specific miRNAs weakens their coupling to AGO2, impairs their function on target mRNAs, determines their delivery into extracellular vesicles (EVs), and provides functional information to receiving cells. Mechanistically, the intracellular functional impairment is caused by m6A-mediated inhibition of AGO2/miRNA interaction, the EV loading is favored by m6A-mediated recognition by the RNA-binding protein (RBP) hnRNPA2B1, and the EV-miRNA function in the receiving cell requires their FTO-mediated demethylation. Consequently, cells express specific miRNAs that do not impact endogenous transcripts but provide regulatory information for cell-to-cell communication. This highlights that a further level of complexity should be considered when relating cellular dynamics to specific miRNAs.

Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disease (IBD) characterized by colorectal inflammation. The N6-methyladenosine (m6A) modification of RNA regulates gene expression through the modulation of RNA metabolism, thus influencing various physiological and pathological processes. The aim of this study was to investigate the biological function of m6A methyltransferase METTL14 in colorectal epithelial cell inflammation. Bioinformatics analysis indicated that METTL14 expression was decreased in UC and was associated with disease severity and immune infiltration. We also noted a downregulation of METTL14 expression and a decrease in the total m6A RNA levels in TNF-α-stimulated Caco-2 cells. Moreover, METTL14 knockdown promoted inflammation and inhibited autophagy in TNF-α-stimulated Caco-2 cells, as indicated by the upregulation of NF-κB signaling and pro-inflammatory cytokine expression as well as LC3B protein downregulation. Treatment with the autophagy activator Torin-1 ameliorated the pro-inflammatory effects of METTL14 silencing. Furthermore, METTL14 knockdown significantly reduced the expression of ATG5. ATG5 overexpression could nullify the pro-inflammatory effect of METTL14 knockdown in TNF-α-stimulated Caco-2 cells. Mechanistically, METTL14 knockdown promoted ATG5 mRNA degradation, and luciferase analysis identified ATG5 as a target of m6A modification by METTL14. Taken together, silencing METTL14 promoted inflammation in Caco-2 cells via the downregulation of ATG5. Our findings revealed the importance of the m6A modification in colonic inflammation and autophagy, indicating that targeting METTL14 might be a potential therapeutic strategy for anti-inflammatory treatment in UC.

Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA-binding protein that is involved in various biological functions, including DNA damage repair and transcription regulation. It plays a crucial role in cisplatin resistance. Nevertheless, the exact regulatory pathways governing PARP1 have not yet been fully elucidated. In this study, we present evidence suggesting that the hepatitis B X-interacting protein (HBXIP) may exert regulatory control over PARP1. HBXIP functions as a transcriptional coactivator and is positively associated with PARP1 expression in tissues obtained from hepatoma patients in clinical settings, and its high expression promotes cisplatin resistance in hepatoma. We discovered that the oncogene HBXIP increases the level of PARP1 m6A modification by upregulating the RNA methyltransferase WTAP, leading to the accumulation of the PARP1 protein. In this process, on the one hand, HBXIP jointly activates the transcription factor ETV5, promoting the activation of the WTAP promoter and further facilitating the promotion of the m6A modification of PARP1 by WTAP methyltransferase, enhancing the RNA stability of PARP1. On the other hand, HBXIP can also jointly activate the transcription factor CEBPA, enhance the activity of the PARP1 promoter, and promote the upregulation of PARP1 expression, ultimately leading to enhanced DNA damage repair capability and promoting cisplatin resistance in hepatoma. Notably, aspirin inhibits HBXIP, thereby reducing the expression of PARP1. Overall, our research revealed a novel mechanism for increasing PARP1 abundance, and aspirin therapy could overcome cisplatin resistance in hepatoma.