Epitranscriptomic Modifications Research Articles

Structural Analysis of Virus Regulatory N6-Methyladenosine (m6A) Machinery of the Black Flying Fox (Pteropus alecto) and the Egyptian Fruit Bat (Rousettus aegyptiacus) Shows Evolutionary Conservation Amongst Mammals

Background: N6-methyladenosine (m6A) is an abundant RNA epitranscriptomic modification in eukaryotes. The m6A machinery includes cellular writer, eraser and reader proteins that regulate m6A. Pteropus alecto (P. alecto) (the Australian black flying fox) and Rousettus aegyptiacus (R. aegyptiacus) (the Egyptian fruit bat) are bats associated with several viral zoonoses yet neglected in the field of m6A epigenetics studies. Objectives: This study utilises various bioinformatics and in silico tools to genetically identify, characterise and annotate the m6A machinery in P. alecto and R. aegyptiacus. Methods: A range of bioinformatic tools were deployed to comprehensively characterise all known m6A-associated proteins of P. alecto and R. aegyptiacus. Results: Phylogenetically, the m6A fat mass and obesity-associated protein (FTO) eraser placed the order Chiroptera (an order including all bat species) in a separate clade. Additionally, it showed the lowest identity matrices in P. alecto and R. aegyptiacus when compared to other mammals (74.1% and 72.8%) and Homo sapiens (84.0% and 76.1%), respectively. When compared to humans, genetic loci-based analysis of P. alecto and R. aegyptiacus showed syntenic conservation in multiple flanking genes of 8 out the 10 m6A-associated genes. Furthermore, amino acid alignment and protein tertiary structure of the two bats’ m6A machinery demonstrated conservation in the writers but not in erasers and readers, compared to humans. Conclusions: These studies provide foundational annotation and genetic characterisation of m6A machinery in two important species of bats which can be exploited to study bat–virus interactions at the interface of epitranscriptomics.

Cannabinoids shift the basal ganglia miRNA m6A methylation profile towards an anti-inflammatory phenotype in SIV-infected Rhesus macaques.

Epitranscriptomic modifications modulate diverse biological processes, such as regulation of gene expression, abundance, location and function. In particular, N6-methyladenosine (m6A) methylation has been shown to regulate various disease processes, including cancer and inflammation. While there is evidence that m6A modification is functionally relevant in neural development and differentiation, the role of m6A modification in HIV neuropathogenesis is unknown. Here, we identified direct m6A modifications in miRNAs from BG tissues of Rhesus Monkeys (RMs) that were either vehicle-treated uninfected (VEH), SIV-infected combination anti-retroviral therapy (cART) treated (VEH/SIV/cART), or THC:CBD treated VEH/SIV/cART (THC:CBD/SIV/cART) RMs. We detected m6A modifications across all BG tissues. SIV infection promoted an overall hypomethylated m6A profile. While the overall hypomethylated m6A profile was not significantly impacted by THC:CBD treatment, specific miRNAs, particularly those predicted to target proinflammatory genes showed markedly reduced m6A methylation levels compared to the VEH treated RMs. Additionally, we found that specific BG tissue miRNAs bearing m6A epi-transcriptomic marks were also transferred to BG-derived extracellular vesicles (EVs). Mechanistically, we identified the DRACH motif of the seed region of miR-194-5p to be significantly m6A hypomethylated, which was predicted to directly target STAT1, an important interferon-activated transcription factor known to drive neuroinflammation, in diseases ranging from Alzheimer to Parkinson and Huntington disease. Notably, THC:CBD treatments significantly reduced m6A methylation of 43 miRNA species directly involved in regulating CNS network genes, thus providing a possible mechanist explanation on the beneficial effects of THC:CBD treatments noted in several disease involving neuroinflammation. Our findings also underscore the need for investigating the qualitative, posttranscriptional modification changes in the RNA profiles along with the more traditional, qualitative alterations in pathological conditions or after various treatment regimens.

Epitranscriptomic m6A modifications during reactivation of HIV-1 latency in CD4+ T cells.

RNA m6A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m6A modification during HIV-1 latency reactivation is unknown. To address this important question, in this study, we used established cellular models of HIV-1 latency, m6A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m6A modification, correlates with cellular m6A levels, and is dependent on the catalytic activity of the m6A methyltransferase enzyme. We also identified cellular genes that are differentially m6A-modified during HIV-1 reactivation, as well as the sites of m6A within HIV-1 RNA. Our novel findings point toward a significant role for m6A modification in HIV-1 latency reversal.

Epigenetic regulation of cardiovascular diseases induced by behavioral and environmental risk factors: Mechanistic, diagnostic, and therapeutic insights

AbstractBehavioral and environmental risk factors are critical in the development and progression of cardiovascular disease (CVD). Understanding the molecular mechanisms underlying these risk factors will offer valuable insights for targeted preventive and therapeutic strategies. Epigenetic modifications, including DNA methylation, histone modifications, chromatin remodeling, noncoding RNA (ncRNA) expression, and epitranscriptomic modifications, have emerged as key mediators connecting behavioral and environmental risk factors to CVD risk and progression. These epigenetic alterations can profoundly impact on cardiovascular health and susceptibility to CVD by influencing cellular processes, development, and disease risk over an individual's lifetime and potentially across generations. This review examines how behavioral and environmental risk factors affect CVD risk and health outcomes through epigenetic regulation. We review the epigenetic effects of major behavioral risk factors (such as smoking, alcohol consumption, physical inactivity, unhealthy diet, and obesity) and environmental risk factors (including air and noise pollution) in the context of CVD pathogenesis. Additionally, we explore epigenetic biomarkers, considering their role as causal or surrogate indicators, and discuss epigenetic therapeutics targeting the mechanisms through which these risk factors contribute to CVD. We also address future research directions and challenges in leveraging epigenetic insights to reduce the burden of CVD related to behavioral and environmental factors and improve public health outcomes. This review aims to provide a comprehensive understanding of behavioral and environmental epigenetics in CVD and offer valuable strategies for therapeutic intervention.

Emerging roles of bases modifications and DNA repair proteins in onco-miRNA processing: novel insights in cancer biology.

Onco-microRNAs (onco-miRNAs) are essential players in the post-transcriptional regulation of gene expression and exert a crucial role in tumorigenesis. Novel information about the epitranscriptomic modifications, involved in onco-miRNAs biogenesis, and in the modulation of their interplay with regulatory factors responsible for their processing and sorting are emerging. In this review, we highlight the contribution of bases modifications, sequence motifs, and secondary structures on miRNAs processing and sorting. We focus on several modes of action of RNA binding proteins (RBPs) on these processes. Moreover, we describe the new emerging scenario that shows an unexpected though essential role of selected DNA repair proteins in actively participating in these events, highlighting the original intervention represented by the non-canonical functions of Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), a central player in Base Excision Repair (BER) pathway of DNA lesions. Taking advantage of this new knowledge will help in prospecting new cancer diagnostic and therapeutic strategies.

M6A RNA methylation controls salivary gland epithelial cell function and has a protective role in Sjögren’s disease

ObjectivesThe RNA epitranscriptomic modification known as N6-methyladenosine (m6A) represents a novel mechanism of gene regulation that is poorly understood in human autoimmune diseases. Our research explores the role of this...

Methyltransferase Like-3-Mediated N6-Methyladenosine Modification of Long Noncoding RNA Hepatocyte Nuclear Factor 1a Antisense RNA 1/Hepatocyte Nuclear Factor 4a Antisense RNA 1 Regulates Cytochrome P450 Enzyme Expression.

Interindividual variations in the expression and activity of cytochrome P450 enzymes (CYPs) led to lower therapeutic efficacy or adverse drug events. We previously demonstrated that CYPs are regulated by the long noncoding RNAs (lncRNAs) hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and HNF4A-AS1 via transcription factors (TFs) including hepatocyte nuclear factor 1a (HNF1A), hepatocyte nuclear factor 4a (HNF4A), and pregnane X receptor (PXR). However, the upstream mechanisms regulating HNF1A-AS1 and HNF4A-AS1 are poorly understood. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mammalian RNA. Therefore, the aim of this study was to investigate whether m6A modification regulates the expression of HNF1A-AS1 and HNF4A-AS1 and affects CYP expression in HepG2 and Huh7 cells. The methyltransferase-like 3 (METTL3) inhibitor, STM2457, significantly suppressed the expression of HNF1A-AS1 and induced HNF4A-AS1 expression. Consistent with this, a loss-of-function assay of METTL3 in the cell lines resulted in the downregulation of HNF1A-AS1 and its downstream HNF1A, PXR, and CYPs at the RNA level, as well as the downregulation of some CYPs proteins, and upregulation of HNF4A-AS1. The results of gain-of-function experiments showed the opposite trend. Mechanistically, subsequent RNA stability experiments confirmed that METTL3 affected the stability of both lncRNAs, but in opposite ways; that is, METTL3 reduced HNF1A-AS1 stability and increased HNF4A-AS1 stability. Rescue experiments confirmed that the regulation of METTL3 on TFs and CYPs may require the involvement of these two lncRNAs. Altogether, our study demonstrates that METTL3 is involved in TFs-mediated CYP expression by affecting HNF1A-AS1/HNF4A-AS1 stability. SIGNIFICANCE STATEMENT: Although the impact of long noncoding RNAs (lncRNAs) including hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and hepatocyte nuclear factor 4a antisense RNA 1 (HNF4A-AS1) on the downstream transcription factor (TF) and cytochrome P450 enzyme (CYP) expression is well studied, the upstream regulation of these two lncRNAs by methyltransferase-like 3 (METTL3) remains unexplored. This study reveals that METTL3 is involved in the regulation of lncRNA-TF-CYP expression by affecting the stability of HNF1A-AS1 and HNF4A-AS1 in HepG2 and Huh7 cells.

Epitranscriptomics and epigenetics: two sides of the same coin?

Gene expression is an intricate biological process that bridges gap between the genotype and the phenotype. Canonical and hereditable epigenetic mechanisms, such as histone and DNA modifications, regulate the release of genetic information encoded in DNA without altering the underlying sequence. Many other non-canonical players, such as chromatin regulators and noncoding RNAs, are also involved in regulating gene expression. Recently, RNA modifications (epitranscriptomics) have been shown to hold enormous potential in shaping cellular transcriptomes. However, their co-transcriptional nature and uncertain heritability mean that they fall outside the current definition of epigenetics, sparking an ongoing debate in the field. Here we will discuss the relationship between canonical and non-canonical epigenetic mechanisms that govern gene expression and offer our perspective on whether (or not) epitranscriptomic modifications can be classified as epigenetic mechanisms.

Chronic social isolation leads to abnormal behavior in male mice through the hippocampal METTL14 mediated epitranscriptomic RNA m6A modifications

BackgroundSocial isolation not only increases the risk of mortality in later life but also causes depressive symptoms, cognitive and physical disabilities. Although RNA m6A modifications are suggested to play key roles in brain development, neuronal signaling and neurological disorders, both the roles of m6A and the enzymes that regulate RNA m6A modification in social isolation induced abnormal behavior is unknown. The present study aims to explore the possible epitranscriptomic role of RNA m6A modifications and its enzymes in social isolation induced impaired behavior. Methods3–4 weeks mice experiencing 8 weeks social isolation stress (SI) were used in the present study. We quantified m6A levels in brain regions related to mood and cognitive behavior. And the expression of hippocampal m6A enzymes was also determined. The role of hippocampal m6A and its enzymes in SI induced abnormal behavior was further verified by the virus tool. ResultsSI led to not only depressive and anxiety-like behaviors but also cognitive impairment, with corresponding decreases in hippocampal m6A and METTL14. Hippocampal over-expression METTL14 with lentivirus not only rescued these behaviors but also enhanced the hippocampal m6A level. Hippocampal over-expression METTL14 resulted in increased synaptic related genes. ConclusionsWe provide the first evidence that post-weaning social isolation reduces hippocampal m6A level and causes altered expression of m6A enzyme in mice. Importantly, hippocampal METTL14 over-expression alleviated the SI-induced depression/anxiety-like and impaired cognitive behaviors and enhanced m6A level and synaptic related genes expression.

Ribosome Biogenesis and Cancer: Insights into NOB1 and PNO1 Mechanisms.

Post-transcriptional modifications (PTMs) are pivotal in the regulation of gene expression, and pseudouridylation is emerging as a critical player. This modification, facilitated by enzymes such as NOB1 (PNO1), is integral to ribosome biogenesis. PNO1, in collaboration with the NIN1/RPN12 binding protein 1 homolog (NOB1), is vital for the maturation of ribosomes, transitioning 20S pre-rRNA into functional 18S rRNA. Recent studies have highlighted PNO1's potential involvement in cancer progression; however, its underlying mechanisms remain unclear. Relentless growth characterizing cancer underscores the burgeoning significance of epitranscriptomic modifications, including pseudouridylation, in oncogenesis. Given PNO1's emerging role, it is imperative to delineate its contribution to cancer development to identify novel therapeutic interventions. This review summarizes the current literature regarding the role of PNO1 in cancer progression and its molecular underpinnings in oncogenesis. Overexpression of PNO1 was associated with unfavorable prognosis and increased tumor malignancy. At the molecular level, PNO1 facilitates cancer progression by modulating mRNA stability, alternative splicing, and translation efficiency. Its role in pseudouridylation of oncogenic and tumor-suppressor transcripts further underscores its significance in cancer biology. Although disruption of ribosome biogenesis is known to precipitate oncogenesis, the precise mechanisms by which these alterations contribute to cancer remain unclear. This review elucidates the intricate process of ribosomal small subunit maturation, highlighting the roles of crucial ribosomal proteins (RPs) and RNA-binding proteins (RBPs) as well as the positioning and function of NOB1 and PNO1 within the 40S subunit. The involvement of these components in the maturation of the small subunit (SSU) and their significance in the context of cancer therapeutics has been thoroughly explored. PNO1's burgeoning significance in oncology makes it a potential target for cancer therapies. Strategies aimed at modulating PNO1-mediated pseudouridylation may provide new avenues for cancer treatment. However, further research is essential to unravel the complete spectrum of PNO1 mechanisms in cancer and harness this knowledge for the development of targeted and more efficacious anticancer therapies.

The m6A methyltransferase METTL14 promotes cell proliferation via SETBP1-mediated activation of PI3K-AKT signaling pathway in myelodysplastic neoplasms

N6-methyladenosine (m6A) is the most prevalent epitranscriptomic modification in mammalian mRNA. Recent studies have revealed m6A is involved in the pathogenesis of various malignant tumors including hematologic neoplasms. Nevertheless, the specific roles of m6A modification and m6A regulators in myelodysplastic neoplasms (MDS) remain poorly understood. Herein, we demonstrated that m6A level and the expression of m6A methyltransferase METTL14 were elevated in MDS patients with bone marrow blasts ≥5%. Additionally, m6A level and METTL14 expression were upregulated as the disease risk increased and significantly associated with adverse clinical outcomes. Knockdown of METTL14 inhibited cell proliferation and colony formation ability of MDS cells. Moreover, in vivo experiments showed METTL14 knockdown remarkably reduced tumor burden and prolonged the survival of mice. Mechanistically, METTL14 facilitated the m6A modification of SETBP1 mRNA by formation of METTL3-METTL14 complex, leading to increased stabilization of SETBP1 mRNA and subsequent activation of the PI3K-AKT signaling pathway. Overall, this study elucidated the involvement of the METTL14/m6A/SETBP1/PI3K-AKT signaling axis in MDS, highlighting the therapeutic potential of targeting METTL3-METTL14 complex-mediated m6A modification for MDS therapy.

A comprehensive review on epigenetic and epitranscriptomic-mediated regulation of antibiotic resistance

Antibiotic resistance is the leading cause of death globally, with a higher possibility of the emergence of highly resistant pathogens, leading to epidemics. Several antibiotic resistance mechanisms have been discovered, such as enhanced efflux of antibiotics, reduced influx of antibiotics, alteration of antibiotics or their targets, and adaptation to antibiotics. However, this mechanism cannot fully explain the development of antibiotic resistance because the genes associated with this mechanism have been elucidated. However, the factors governing their regulation are not yet fully understood. Recent studies have highlighted the epigenetic and epitranscriptomic roles of antibiotic resistance development-associated genes. Epigenetic modification is associated with DNA modification, whereas epitranscriptomic modification is associated with RNA modification to control gene expression by regulating various biological phenomena such as splicing, translation, and stability. Therefore, this review will focus on the discovery of epigenetic modifications, particularly by DNA methyltransferases, such as restriction-modification (R-M) systems associated with methyltransferases, orphan DNA methyltransferases, and nucleoid-associated proteins that contribute to the development of antibiotic resistance. This scrutinization further expands to epitranscriptomic modification of non-coding RNA, which has a role in the regulation of antibiotic resistance. Epitranscriptomic modification of ribosomal RNA (rRNA), which is a major target of antibiotics, has been well explored. while non-coding RNA such as cis and trans small non coding RNA, and riboswitches are poorly explored. This epigenetic and epitranscriptomic modification will help to understand the regulation of antibiotic resistance-associated genes, which will help to identify key regulators of antibiotic resistance, paving the way for new antibiotic discovery, leading to decreased antibiotic mortality globally.

Regulatory RNAs: role as scaffolds assembling protein complexes and their epigenetic deregulation.

Recently, new data have been added to the interaction between non-coding RNAs (ncRNAs) and epigenetic machinery. Epigenetics includes enzymes involved in DNA methylation, histone modifications, and RNA modifications, and mechanisms underlying chromatin structure, repressive states, and active states operating in transcription. The main focus is on long ncRNAs (lncRNAs) acting as scaffolds to assemble protein complexes. This review does not cover RNA's role in sponging microRNAs, or decoy functions. Several lncRNAs were shown to regulate chromatin activation and repression by interacting with Polycomb repressive complexes and mixed-lineage leukemia (MLL) activating complexes. Various groups reported on enhancer of zeste homolog 2 (EZH2) interactions with regulatory RNAs. Knowledge of the function of these complexes opens the perspective to develop new therapeutics for cancer treatment. Lastly, the interplay between lncRNAs and epitranscriptomic modifications in cancers paves the way for new targets in cancer therapy. The approach to inhibit lncRNAs interaction with protein complexes and perspective to regulate epitrascriptomics-regulated RNAs may bring new compounds as therapeuticals in various types of cancer.

Crosstalk between epitranscriptomic and epigenomic modifications and its implication in human diseases

Crosstalk between N6-methyladenosine (m6A) and epigenomes is crucial for gene regulation, but its regulatory directionality and disease significance remain unclear. Here, we utilize quantitative trait loci (QTLs) as genetic instruments to delineate directional maps of crosstalk between m6A and two epigenomic traits, DNA methylation (DNAme) and H3K27ac. We identify 47 m6A-to-H3K27ac and 4,733 m6A-to-DNAme and, in the reverse direction, 106 H3K27ac-to-m6A and 61,775 DNAme-to-m6A regulatory loci, with differential genomic location preference observed for different regulatory directions. Integrating these maps with complex diseases, we prioritize 20 genome-wide association study (GWAS) loci for neuroticism, depression, and narcolepsy in brain; 1,767 variants for asthma and expiratory flow traits in lung; and 249 for coronary artery disease, blood pressure, and pulse rate in muscle. This study establishes disease regulatory paths, such as rs3768410-DNAme-m6A-asthma and rs56104944-m6A-DNAme-hypertension, uncovering locus-specific crosstalk between m6A and epigenomic layers and offering insights into regulatory circuits underlying human diseases.

Stroke triggers dynamic m6A reprogramming of cerebral circular RNAs

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N6-methyladenosine (m6A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m6A methylation of circRNAs. Changes in m6A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m6A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m6A modification patterns. The majority of circRNAs that showed post-stroke differential m6A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m6A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m6A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology.

Plasmodium falciparum artemisinin resistance: something gained in translation

Small-Saunders et al. uncovered a new facet of artemisinin resistance in Plasmodium in which parasites use a previously underexplored arm of stress response mechanisms. Through altered epitranscriptomic modifications on tRNA, changed translation patterns adapt resistant cells to facilitate entry into a quiescent-like state which provides the parasite an escape from many drugs.

Emerging Role of Environmental Epitranscriptomics and RNA Modifications in Parkinson's Disease.

Environmental risk factors and gene-environment interactions play a critical role in Parkinson's disease (PD). However, the relatively large contribution of environmental risk factors in the overwhelming majority of PD cases has been widely neglected in the field. A "PD prevention agenda" proposed in this journal laid out a set of research priorities focused on preventing PD through modification of environmental risk factors. This agenda includes a call for preclinical studies to employ new high-throughput methods for analyzing transcriptomics and epigenomics to provide a deeper understanding of the effects of exposures linked to PD. Here, we focus on epitranscriptomics as a novel area of research with the potential to add to our understanding of the interplay between genes and environmental exposures in PD. Both epigenetics and epitranscriptomics have been recognized as potential mediators of the complex relationship between genes, environment, and disease. Multiple studies have identified epigenetic alterations, such as DNA methylation, associated with PD and PD-related exposures in human studies and preclinical models. In addition, recent technological advancements have made it possible to study epitranscriptomic RNA modifications, such as RNA N6-methyladenosine (m6A), and a handful of recent studies have begun to explore epitranscriptomics in PD-relevant exposure models. Continued exploration of epitranscriptomic mechanisms in environmentally relevant PD models offers the opportunity to identify biomarkers, pre-degenerative changes that precede symptom onset, and potential mitigation strategies for disease prevention and treatment.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Dynamic RNA methylation modifications and their regulatory role in mammalian development and diseases.

Among over 170 different types of chemical modifications on RNA nucleobases identified so far, RNA methylation is the major type of epitranscriptomic modifications existing on almost all types of RNAs, and has been demonstrated to participate in the entire process of RNA metabolism, including transcription, pre-mRNA alternative splicing and maturation, mRNA nucleus export, mRNA degradation and stabilization, mRNA translation. Attributing to the development of high-throughput detection technologies and the identification of both dynamic regulators and recognition proteins, mechanisms of RNA methylation modification in regulating the normal development of the organism as well as various disease occurrence and developmental abnormalities upon RNA methylation dysregulation have become increasingly clear. Here, we particularly focus on three types of RNA methylations: N6-methylcytosine (m6A), 5-methylcytosine (m5C), and N7-methyladenosine (m7G). We summarize the elements related to their dynamic installment and removal, specific binding proteins, and the development of high-throughput detection technologies. Then, for a comprehensive understanding of their biological significance, we also overview the latest knowledge on the underlying mechanisms and key roles of these three mRNA methylation modifications in gametogenesis, embryonic development, immune system development, as well as disease and tumor progression.

METTL8 links mt-tRNA m3C modification to the HIF1α/RTK/Akt axis to sustain GBM stemness and tumorigenicity

Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.